Chlamydophila pneumoniae HflX belongs to an uncharacterized family of conserved GTPases and associates with the Escherichia coli 50S large ribosomal subunit.

Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [gamma-(32)P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

Serum-free culturing of mammalian cells--adaptation to and cryopreservation in fully defined media.

Long term storage of living cells is a central issue in cell biology and medicine. In addition to the cryoprotectant dimethyl sulphoxide (DMSO), foetal bovine serum (FBS) is often added to the freezing medium for the cryoconservation of serum dependent cell lines. FBS, with its high protein content, protects cells against shear forces and gives the medium a desirable osmotic environment with a physiological viscosity. However, the harvesting of FBS is painful for the foetus and should be avoided for ethical reasons. In this work we describe the adaptation of several commonly used cell lines to serum- and protein-free media; however, such cell lines should not be frozen in a conservation medium containing serum. We tested the synthetic surfactant ''Pluronic F68'', known to protect mammalian cells grown in serum-free bioreactors (Papoutsakis, 1991), as an active cryoprotectant. In samples containing 0.1 to 1% Pluronic F68, we found a significant increase in viable cells after thawing. Values up to 115% of starting cell number indicate that the cells proliferate within the first 24 hours after thawing, a property which was not observed in cryoconservation media without Pluronic F68.

Genotoxic effect of ozone in human peripheral blood leukocytes.

The genotoxic effect of ozone was studied in human leukocytes in vitro, using the single cell gel electrophoresis (SCGE) assay. Cell treatment for 1 h at 37 degrees C with 0.9-5.3 mM O(3) resulted in a dose-dependent increase of DNA damage, comparable to that induced by 4-40 mM of H(2)O(2), used as a positive control. This effect of ozone was reversed by post-treatment incubation of the cells for 45-90 min at 37 degrees C, and prevented by pre-incubation of the cells with catalase (20 microg/ml). These results demonstrate that O(3) induces DNA-damage in primary human leukocytes. The damage is rapidly repaired, and probably mediated by the formation of H(2)O(2).

Estudio de pacientes sometidos a ozonoterapia mediante el ensayo de electroforesis alcalina de células individuales / Study of patients undergoing ozone therapy by the alkaline electrophoresis test of individual cells

El ozono es un poderoso agente oxidante. Sin embargo, en predeterminadas condiciones y concentraciones es aplicado en enfermedades que cursan con un déficit en las defensas antioxidantes con el objetivo de estimularlas. Para el estudio del efecto genotóxico del ozono gaseoso utilizado en la autohemoterapia se estableció un diseño experimental el cual fue el realizado en 3 pacientes tratados con 200 mL de O3/O2(50 mg/mL) vía autohemoterapia y 1 paciente tratado vía rectal con la misma dosis y concentración. Se tomaron 0,5 ml de sangre antes y después del tratamiento en los días 1, 7 y 14 y se manifestó un incremento del daño en los días 1 y 14 de la terapia después del tratamiento, no ocurriendo así en el día 7, donde se reflejó un posible período de adaptación. En ambos casos el análisis de las muestras se llevó a cabo utilizando el ensayo alcalino de electroforesis de células individuales o ensayo cometa que evalúa las rupturas de simple cadena y los sitios labiles al álcali en el ADN(AU)