An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

Variability of 6 Colombian strains of Trypanosoma cruzi with restriction fragment length polymorphisms (RFLP) and random amplification of polymorphic DNA (RAPD)

Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a public health problem in Colombia. Previous reports have indicated the presence of heterogeneity among parasite populations. Six Colombian T. cruzi strains were obtained that differed by host, geographical region and transmission cycle. The genetic variability of each was compared by random amplified polymorphic DNA (RAPD), and isoenzymes. A restriction fragment length polymorphism (RFLP) was extracted using the 1.2 kb unit encoding the parasite's H2A histone as a probe. Genetic distances between the isolates varied greatly, from 0.611 to 0.99 as determined by RAPD profiles (M13F and M13R primers), between 0 and 0.81 by RFLP profiles (5 endonucleases), and between 0.10 and 0.55 by isoenzymes (13 enzymatic systems). Genetic distance matrixes derived from each of the three methods showed that Colombian strains exhibit a high degree of genetic differentiation. This may account for the broad clinical spectrum of Chagas disease in Colombia.     

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.     

Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor,a static-cell bioreactor and static cell cultures

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2). Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.     

Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.     

Alpha-helix structure in Alzheimer's disease aggregates of tau-protein

The discovery of beta-sheet structure in Alzheimer's amyloid fibrils, and then in many other disease-related protein fibrils, has led to the widely believed view that beta-sheet formation is the general mechanism of aberrant protein aggregation leading to disease. This notion is further reinforced by recent findings, which indicate that normal proteins can be induced to form beta-sheet fibrils in vitro. Alzheimer's disease, a paradigm proteopathy, is accompanied by the formation of two distinct aggregates, amyloid fibrils and paired helical filaments (PHFs). Electron microscope images of PHFs show pairs of twisted ribbons with 80 nm periodicity. However, there is little information of the molecular structure of PHFs, as previous studies have failed to identify signs of regular structure. Using far-UV circular dichroism and Fourier-transformed infrared spectroscopy, we find that PHFs are comprised of alpha-helices. This is remarkable as tau-protein, PHF's primary constituent, has a high abundance of helix-breaking amino acids and is unstructured in solution. We also find that PHFs are very stable, as judged by their high melting temperature and resistance to protease digestion. PHFs are the first example of pathological aggregation associated to the formation of alpha-helix.     

Location and properties of the taxol binding center in microtubules: a picosecond laser study with fluorescent taxoids

The interaction of two bioactive, fluorescent analogues of the anticancer drug Taxol, Flutax1 [7-O-[N-(fluorescein-4'-carbonyl)-L-alanyl]taxol] and Flutax2 [7-O-[N-(2,7-difluorofluorescein-4'-carbonyl)-L-alanyl]taxol], with microtubules in solution has been studied with picosecond laser methods. As shown here, although a mixture of the fluorescein mono- and dianion species of Flutax1 is present in solution, the bound taxoid contains only the dianion form of the dye. This indicates strong electrostatic interactions at the microtubule lattice with the appending dye, most likely with charged residues of the M-loop of the beta-tubulin subunit. Moreover, analysis of the dynamic depolarization of microtubule-bound Flutax at low binding site occupancy was consistent with a protein active center with significant conformational flexibility. On the other hand, for microtubules fully saturated with the taxoid, a new, additional depolarizing process was observed, with relaxation times of 14 ns (Flutax1) and 8 ns (Flutax2), which is due to F?rster resonance energy homotransfer (FREHT) between neighboring dye molecules. Application of a detailed analysis of FREHT-induced depolarization in a circular array of dye molecules presented here yielded a separation between nearest-neighbor Flutax moieties of 40 +/- 5 A, for microtubules made up of between 12 and 14 protofilaments, a value that is only compatible with the Taxol binding site being located at the inner wall of the microtubule. The internal position of the drug molecular target as measured here is also consistent with other spectroscopic observations and confirms existing predictions based on microtubule structures modeled from high-resolution, electron density maps of alphabeta-tubulin.