Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10⁹ BHK-21 cells from 4 × 10⁶ cells in 13 day with 1,051 mL culture medium.     

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.     

Identification and partial characterisation of a chitinase from Nile tilapia, Oreochromis niloticus

Measurement of chitinase activity in extracts from stomach, intestine, and serum of Nile tilapia with the artificial substrates 4-methylumbelliferil beta-D-N,N'-diacetylchitobioside and 4-methylumbelliferil beta-D-N,N'N"-triacetylchitotrioside (4MU[GlcNAc](2,3)) showed that an endochitinase was involved in the liberation of the fluorophore 4-methylumbelliferone (MU). Enzymes were isolated from tilapia serum by a combination of gel filtration, ion exchange, and reverse-phase chromatography. The molecular mass of the enzyme was estimated to be 75 kDa by SDS-PAGE, suggesting that the enzyme occurs as a monomer. The partially purified enzyme showed maximal activity at pH 7.0 when assayed with 4MU[GlcNAc](2) and lost its activity below pH 5.0 and above pH 8.0. The optimal pH of the purified enzyme toward the substrate 4MU[GlcNAc](3) was pH 9.0 and activity was lost below pH 8.0 and above pH 9.0. Our study has revealed the presence of a chitinolytic enzyme in the gastrointestinal tract and serum that may play a role in digestion and/or defense.     

Resistance of Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) to acaricides that inhibit cellular respiration in citrus: cross-resistance and fitness cost

Acaricides that inhibit cellular respiration play an important role in the control of Brevipalpus phoenicis (Geijskes) in citrus groves in Brazil. Studies were conducted to evaluate: (a) the variability in the susceptibility among B. phoenicis populations collected from citrus groves to cyhexatin, azocyclotin, propargite and sulphur; (b) cross-resistance relationships between propargite and azocyclotin, cyhexatin, dinocap, pyridaben and sulphur; and (c) the fitness cost associated with propargite resistance in B. phoenicis under laboratory conditions. A residual-type contact bioassay was used to characterize the susceptibility. The susceptibility was estimated with diagnostic concentrations based LC(95) of each acaricide. The cross-resistance was evaluated by characterizing the concentration-mortality responses of susceptible (S) and propargite-resistant (Propargite-R) strains. The fitness cost was evaluated by measuring the biological parameters of S and Propargite-R strains on citrus fruits at 25 +/- 1 degrees C and fotophase of 14h. Significant differences in the susceptibility of B. phoenicis were detected at diagnostic concentration of cyhexatin (survivorship from 16.3% to 80.5%), azocyclotin (from 3.0% to 15.0%), propargite (from 1.0% a 71.6%) and sulphur (from 9.0% to 82.6%). A low intensity of cross-resistance was detected between propargite and the acaricides azocyclotin (1.8-fold), cyhexatin (4.6-fold), dinocap (3.5-fold) and pyridaben (3.5-fold). On the other hand, the intensity of cross-resistance to sulphur (> 111-fold) was very high. There was no fitness cost associated with B. phoenicis resistance to propargite, based on biological parameters evaluated. Therefore, the use of these acaricides should also be done very carefully in resistance management of B. phoenicis to acaricides.     

Stem cells remember their grade

The stem cell state is understood based on what cells do in performance assays, crude measures of a highly refined state. In this issue of Cell Stem Cell, Dykstra et al. (2007) reveal stem cell gradation and the extent to which that gradation is retained in stem cell daughters of hematopoietic stem cells.     

Increases in plasma corticosterone and stretched-attend postures in rats naive and previously exposed to the elevated plus-maze are sensitive to the anxiolytic-like effects of midazolam

A single exposure to the elevated plus-maze test (EPM) reduces or abolishes the anxiolytic efficacy of benzodiazepines on a second trial. This phenomenon known as one-trial tolerance (OTT) is considered to be due to a shift in the emotional state of the animals across the test/retest sessions. Activation of the hypothalamic-pituitary-adrenocortical (HPA) axis has been considered to be an adaptive response to stressful or challenging situations such as height and openness of the EPM. This work looks at the phenomenon of OTT to the benzodiazepine compound midazolam through the conjoint examination of the novel ethological measures of the EPM and adrenocortical response of rats exposed to single and repeated sessions of the EPM. The results obtained confirmed that the approach/avoidance conflict on the first trial of the EPM is very sensitive to the anxiolytic effects of benzodiazepines. Moreover, stressful stimuli present upon initial exposure to the EPM render the standard measures of the EPM resistant to the anxiolytic effects of benzodiazepines on retest. However, the increases in plasma corticosterone and in risk assessment behavior observed in rats submitted to single or repeated sessions in the EPM were reversed by pretreatment with midazolam. The administration of metyrapone, a glucocorticoid synthesis blocker, decreased risk assessment but did not affect locomotion and anxiety-like behaviors. It is suggested that the detection of the dangerous environment through the stretched-attend postures in the second trial leads to the known low level of exploration and the consequent OTT upon retest. Moreover, in view of the similarity between the risk assessment and plasma corticosterone patterns in both naive and experienced rats, this hormone-behavior profile may be crucial for the expression of OTT to benzodiazepines in rodents exposed to the EPM.     

Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress

Proline accumulates in a variety of plant species in response to stresses such as drought, salinity and extreme temperatures. Although its role in plant osmotolerance remains controversial, proline is thought to contribute to osmotic adjustment, detoxification of reactive oxygen species and protection of membrane integrity. In the present study, we evaluated the effects of stress-inducible proline production on osmotic adjustment, chlorophyll fluorescence and oxidative stress protection in transgenic sugarcane transformed with a heterologous P5CS gene. In well-watered conditions, free proline, malondialdehyde (MDA) levels, Fv/Fm ratios and chlorophyll contents (Chls) in transgenic sugarcane were not statistically different from non-transformed control plants. After 9 days without irrigation, proline content in transgenic events was on the average 2.5-fold higher than in controls. However, no osmotic adjustment was observed in plants overproducing proline during the water-deficit period. The photochemical efficiency of PSII observed was higher (65%) in the transgenic events at the end of the water-deficit experiment. The effects of proline on lipid peroxidation as MDA levels and on the decline of Chl in paraquat-treated leaf discs along the drought period suggest that proline protected the plants against the oxidative stress caused by the water deficit. The overall capacity of transgenic plants to tolerate water-deficit stress could be assessed by the significantly higher biomass yields 12 days after withholding water. These results suggest that stress-inducible proline accumulation in transgenic sugarcane plants under water-deficit stress acts as a component of antioxidative defense system rather than as an osmotic adjustment mediator.     

Proceedings of workshop: “Neuroglycoproteins in health and disease”, INNOGLY cost action

Glycoconjugate Journal - The Cost Action “Innovation with glycans: new frontiers from synthesis to new biological targets” (INNOGLY) hosted the Workshop “Neuroglycoproteins in...