Studies on the development of metacyclic Trypanosoma brucei sspp. cultivated at 27 degrees C with insect cell lines

When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T.b. rhodesiense were grown at 27 degrees C in 25-cm2 flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice. Infectivity titrations on trypanosome suspensions revealed that up to 2.8 X 10(5) metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated. Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful. Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections. Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity. Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.     

Infectivity of Trypanosoma rhodesiense Cultivated at 28°C with Various Tsetse Fly Tissues1

ABSTRACT. Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28°C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7–16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUM 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 545, and TRUM 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes ceased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium “conditioned” by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

Infectivity of Trypanosoma brucei Cultivated at 28 C with Tsetse Fly Salivary Glands*

SYNOPSIS. When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 27 and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

Development of metacyclic forms of Trypanosoma brucei sspp. in cultures containing explants of Phormia regina Meigen

When procyclic trypanosomes of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense were cultivated in Nunclon 25 cm2 flasks at 27 C in a liquid medium containing various tissue explants of Phormia regina Meigen, some of them developed into forms infective for mice. The infective stages were present at various periods of up to 29 days when the cultures were terminated. Larger numbers of explants of head-salivary glands than the other tissues used were required to produce infections. Infectivity titrations on trypanosome suspensions of T. b. brucei TRUM 252 and T. b. rhodesiense TRUM 497 indicated that only a small proportion of the populations was infective. Mice were rarely infected with trypanosomes grown in medium without explants. Only 1 mouse of the 11 inoculated developed a parasitemia from a control culture of T. b. rhodesiense TRUM 545. A few trypanosomes resembling epimastigotes and metacyclic forms were seen in stained samples of infective inocula.     

Comparisons of blood viscosity between amphibians and mammals at 3°C and 38°C

(1) The range of temperature exposure of endotherms is narrow compared to ectotherms that can experience daily and seasonal temperature fluxes. (2) Comparison of the blood viscosity of amphibians (bullfrog, Woodhouse's toad, and marine toad) and mammals (horse, dog, and rat) at 3°C and 38°C was undertaken to determine if the effect of temperature on blood viscosity was diminished in amphibians relative to mammals. (3) Mammals did not consistently show greater changes in blood viscosity, plasma viscosity, or relative viscosity with decreasing temperatures relative to the amphibians in this study. (4) These data do not support our hypothesis that blood viscosity of amphibians is less affected by temperature than mammalian blood.     

The Axenic Cultivation of Insect Forms of Trypanosoma (Duttonella) vivax and Development to the Infective Metacyclic Stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27°C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine, hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9–12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.     

Infectivity of Trypanosoma brucei cultivated at 28 C with tsetse fly salivary glands

When transformed procyclic noninfective trypanosomes of several unrelated stocks of Trypanosoma brucei were cultivated in T-30 Falcon flasks at 28 C in a liquid medium containing head-salivary gland explants of Glossina morsitans morsitans some of the organisms developed into forms infective for mice. Infective trypanosomes were detected 7 to 14 days after the cultures were prepared and they persisted for varying periods of up to 88 days when the cultures were terminated. A few of the salivary glands became invaded with parasites about the time infective organisms appeared in the cultures. Using T. brucei TREU 929, it was shown that trypanosomes grown with between 2m and 50 explants were capable of producing infections consistently for prolonged periods. On the other hand, trypanosomes cultivated with 25 or fewer explants rarely infected mice. Infectivity titrations on trypanosome suspensions from cultures of stocks TREU 1275 and TREU 929 revealed that the maximum number of infective organisms was present 26 to 50 days after initiation of the cultures. Control cultures of trypanosomes grown in medium alone were generally not infective but 2 of the 6 stocks gave rise to a few sporadic infections. A few epimastigote-like and metacyclic-like trypanosomes were seen in stained preparations of infective inocula.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Induction and Rejoining of DNA Single-Strand Breaks in Relation to Cellular Growth in Human Cells Exposed to Three Hydroperoxides at 0°C and 37°C

There was a 5-fold increase in cytotoxicity for cumene hydroperoxide, 10-fold for tert-butyl hydroperoxide and 25-fold for hydrogen peroxide, under metabolizing conditions (37°C) in comparison to nonmetabolizing conditions (0°C), when human P31 cells were exposed for 60 min. The induction of DNA single-strand breaks correlated poorly with cytotoxicity. Hydrogen peroxide was by far the most effective agent inducing single-strand breaks irrespective of temperature. Cumene hydroperoxide produced fewer strand breaks than tert-butyl hydroperoxide despite its greater cytotoxicity at either 37°C or at 0°C. The pattern of single-strand break induction did not change with temperature. The number of breaks, however, increased when the cells were exposed at 37°C. The pattern of rejoining was similar for hydrogen peroxide- and tert-butyl hydroperoxide-induced breaks at both temperatures whereas the rejoining of cumene hydroperoxide-induced breaks deviated somewhat from this pattern. The results indicate that there is no clear-cut relationship between induction of DNA single-strand breaks and cytotoxicity after hydroperoxide exposure.     

Improved fluorometric assay of histamine: Condensation with O-phthalaldehyde at −20°C

Histamine reacts with OPT at an alkaline pH giving rise to fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 10 hr under nitrogen at −20°C, i.e., in the frozen state. After acidification with sulfuric acid to pH 2.5 the resulting fluorescence, read at room temperature, was stable for hours. The procedure now measures as little as 1 ng histamine/ml and is much more specific than the conventional fluorometric assay. Spermidine did not interfere with the assay of histamine, and histidine only if present in great excess over histamine. It could be shown that with deproteinized extracts of rat gastric mucosa, histamine could be estimated without further purification, which means saving a lot of time and labor.     

Free Radicals Appear to Affect Survival of Hepatocytes at 4°C

1) Rat hepatocytes, stored in a simple salts medium for 24 h at 4°C, retain more than 80% of their capacity to synthesize glucose from lactate.

2) The combination of NH₄Cl with oleate is cytotoxic during storage and during subsequent incubation of hepatocytes from 48 h starved rats, but not to hepatocytes from fed rats.

3) Protection against cytotoxicity is afforded by albumin and by a number of other compounds, notably polyols and glycerol.

4) These compounds appear to exert their effects by scavenging free radicals and, in the case of polyols and glycerol, by supplying reducing equivalents to maintain the redox state of the cell in the face of increased flux through glutathione peroxidase.     

Cell Adhesion in Trypanosoma: In Vitro Studies of the Interaction of Trypanosoma vivax with Immobilized Organic Dyes1

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex? Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.     

Mechanical properties of the human red blood cell membrane at −15 °C

The most common method for measuring the mechanical behavior of the human red blood cell (RBC) membrane is micropipette aspiration, because it can be used to apply both a low uniaxial stress at a small part of the membrane or high two-axial stresses to the whole membrane [E.A. Evans, R.E. Waugh, Mechano-chemical study of red cell membrane structure in situ, in: Kroc Foundation Series, vol. 13, Erythrocyte Mechanics and Blood Flow, Alan R. Liss. Inc., New York, 1980, pp. 31–56 (Chapter 3); H.J. Meiselman, Measures of blood rheology and erythrocyte mechanics, in: Kroc Foundation Series, vol. 13, Erythrocyte Mechanics and Blood Flow, Alan R. Liss. Inc., New York, 1980, pp. 75–117 (Chapter 5)]. The elastic shear moduli and area changes of the human RBC published to date were calculated by means of this technique. However, a main drawback of the method is its impracticability at subzero temperatures. Experiments at below 0 °C are of interest because it is at these temperatures that RBC lysis occurs during freezing and thawing after cryopreservation, via a mechanism that may be mechanical.A method for circumventing this limitation is deforming the cell membranes by applying an electric ac field to a supercooled suspension. In a previous study, we applied this technique to human RBCs down to −15 °C [M. Krueger, F. Thom, Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures, Biophys. J. 73 (1997) 2653–2666]. In this technique, the electrical dimensions must be translated into those of mechanics. We provided a formula for these calculations, which demonstrated excellent concordance with known mechanical measurements at room temperature [F. Thom, H. Gollek, Calculation of mechanical properties of human red cells based on electrically induced deformation experiments, J. Electrostat. 64 (2006) 53–61]. Using this formula, we have now calculated the shear moduli and stress–strain diagram for our deformation experiments at −15 °C and present the results below.     

Thermodynamic interaction between urea and the peptide group in aqueous solutions at 25°C

Isopiestic vapor pressure measurements of the ternary systems water + triglycine + urea and water + glycine-L-alanine + urea were made and used to calculate the Gibbs free energy of these systems. Together with recently published analogous results on systems, in which the first solute was glycine or alanine or diglycine, and measurements of the excess enthalpy of all these solutions, it is possible to calculate the Gibbs free energy of transfer and the enthalpy of transfer of the peptide group from water to aqueous urea solutions. The transfer can be described as a binding of urea to the peptide group with ΔG = ?1.85 kJ mol^?1 and ΔH = ?18.7 kJ mol^?1 at 298.1 K.     

Studies on the recycling of the transferrin receptor in Trypanosoma brucei using an inducible gene expression system.

Uptake of host transferrin in bloodstream forms of Trypanosoma brucei is mediated by a heterodimeric, glycosylphosphatidylinositol-anchored receptor. After endocytosis, transferrin is delivered to lysosomes where it is proteolytically degraded. Whether the heterodimeric transferrin receptor is returned to mediate several cycles in ligand uptake is undefined. By using an inducible gene expression system we provide evidence for recycling of the transferrin receptor in bloodstream forms of T. brucei. The metabolic half-life of the transferrin receptor in bloodstream-form trypanosomes is determined to be 7 h which is comparable to the half-lives of recycling receptors in mammalian cells. The cycling time of the trypanosomal transferrin receptor is calculated to be 11 min. By means of the half-life and the cycling time, we calculated that each receptor is recycled 60 times before being degraded on average.