Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

Background: To reduce costs associated with productivity of recombinant proteins in the biopharmaceutical industry, research has been focused on regulatory principals of growth and survival during the production phases of the cell culture. The main strategies involve the regulation of cell proliferation by the modulation of cell cycle control points (G1/S or G2/M) with mild hypothermia and the addition of sodium butyrate (NaBu). In this study, batch culture strategies were evaluated using CHO TF 70R cells producing the recombinant human tissue plasminogen activator (rh-tPA), to observe their individual and combined effect on the cellular physiological state and relevant kinetic parameters. Results: NaBu addition has a negative effect on the mitochondrial membrane potential (ΔΨm), the values of which are remarkably diminished in cultures exposed to this cytotoxic compound. This effect was not reflected in a loss of cell viability. NaBu and mild hypothermic conditions increased the doubling time in the cell cultures, suggesting that these strategies triggered a general slowing of each cell cycle phase in a different way. Finally, the individual and combined effect of NaBu and mild hypothermia produced an increase in the specific rh-tPA productivity in comparison to the control at 37°C without NaBu. Nevertheless, both strategies did not have a synergistic effect on the specific productivity. Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Measuring b-Galactosidase activity at pH 6 with a differential pH sensor

The b-Galactosidase activity at pH 6 is used as a cellular marker to identify senescent cell cultures. The classic method to identify this enzymatic activity is using cytochemical staining with X-Gal after 16 hrs. In this work, a differential pH sensor was used to measure b-Galactosidase activity at pH 6. The measurement is easy and only takes 3 min.

Volatile Organic Compounds Produced by Human Skin Cells

Skin produces volatile organic compounds (VOCs) released to the environment with emission patterns characteristic of climatic conditions. It could be thought that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, using gas chromatography, we answered the question of whether VOC profiles of primary cultures of human dermal fibroblasts were affected by the type of culture conditions. VOCs were determined for different types of culture, finding significant differences between skin cells grown in classical monolayer culture -2D- compared with 3D matrix immobilized cultures. This indicates that VOC profiles could provide information on the physiological state of skin cells or skin.

A fluorescent method to determine picomole amounts of Zn(II) in biological systems

The formation of complexes between n-(6-methoxy-8-quinolyl)-p-toluensulfonamide (TSQ) and Zn(II) in methanol has been used as an analytical procedure for Zn(II) determination in biological systems. Using content determined by atomic absorption measurements. In rat spermatids, deproteinization and Zn (II) determination using the TSQ method gives approximately a 20 percent underestimation of the total Zn (II) content of the cells as compared to1 ml cuvettes, the limit of detection of the method was approximately 20 pmoles of Zn(II). Linearity between fluorescence and zinc concentration was obtained up to approximately 1 microM Zn(II). Common ltivalent cations present in biological systems like Al3+, Cu2+, Fe3+, Ca2+, and Mg2+, interfered with the measurement of Zn(II) only when present in excess of 20, 33, 60, 500 and 30,000 times the Zn (II) concentration, respectively. In human serum and semen, deproteinization of the samples permitted a goodcorrelation between the TSQ method and the total Zn atomic absorption spectrophotometry. The method gives low resolution of Zn (II) peaks when tested as an analytical procedure to measure Zn (II) binding to protein fractions eluted during column chromatography