Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Studies on the cationization of agarose

Cationized agaroses with different degrees of substitution (0.04–0.77) were synthesized, employing 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC). The influence of different reaction parameters on the substitution degree and molecular weight was evaluated. The investigated parameters were concentration of reagents, temperature, time, and addition of NaBH₄. The products were characterized by means of scanning electronic microscopy, infrared spectroscopy, viscosimetry, and NMR spectroscopy. Methanolysis products were studied by electrospray ionization mass spectrometry. The higher the concentration of CHPTAC employed, a higher degree of substitution was obtained, if the optimum concentration of NaOH in each case was employed. Insufficient quantities of NaOH reduced epoxide formation and the reacting alkoxides of the polysaccharide, whereas an excess of NaOH favored degradation of the epoxide and decrease in the molecular weight of the product. A reaction time of 2 h was sufficient to obtain products with the maximum degree of substitution for each case. The addition of NaBH₄ gave products with a slightly higher molecular weight, but the extra cost involved should not justify its use for large-scale application.     

New secretory strategies for Kluyveromyces lactis beta-galactosidase.

We examined several strategies for the secretion of Kluyveromyces lactis beta-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed plasmids by fusing the LAC4 gene or engineered variants to the secretion signal of the K.lactis killer toxin or to the secretion signal of the Saccharomyces cerevisiae alpha-factor. With these plasmids we transformed strains of the yeasts K.lactis and S.cerevisiae, respectively and tested beta-galactosidase extracellular activity in different culture media. We achieved partial secretion of beta-galactosidase in the culture medium since the high molecular weight and oligomeric nature of the enzyme, among other factors, preclude full secretion. The percentage of secretion was improved by directed mutagenesis of the N-terminus of the protein. We developed several deletion mutants which helped us to propose structure-function relationships by comparison with the available data on the homologous Escherichia coli beta-galactosidase. The influence of the culture conditions on heterologous beta-galactosidase secretion was also studied.     

The D3-creatine dilution method non-invasively measures muscle mass in mice

Developing accurate methods to quantify age-related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual-energy X-ray absorptiometry (DXA) but does not measure muscle directly—it is a composite measure quantifying “lean mass” (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D₃-creatine (D₃Cr) dilution method for quantifying muscle mass was developed in humans and rats. This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D₃Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D₃Cr method compared to DXA-determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D₃Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non-invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.     

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.     

Supersensitivity of the isolated guinea-pig vas deferens induced by a toxic fraction of scorpion venom (Tityus serrulatus)

Tityustoxin (TsTx), a toxic fraction of Tityus serrulatus venom, was studied on the isolated guinea-pig vas deferens. It increased significantly the maximal response of the preparation to both norepinephrine and acetylcholine and decreased the effective median dose of norepinephrine. The effect of TsTx on norepinephrine median dose was unchanged when atropinized or pharmacologically "denervated" preparations were used but was abolished when both procedures were associated. Atropinization of pharmacologically denervated muscles almost never modify the TsTx-induced increase in the maximal response to norepinephrine. On denervated or phentolamine-treated muscles TsTx-induced increase in the maximal response to acetylcholine was abolished. It was concluded that toxin predominantly induces adrenergic postsynaptic supersensitivity. Of minor significance, it also induces presynaptic cholinergic and adrenergic supersensitivity. Comparison of these results with those of crude venom indicates that TsTx effects may result from the sum of the effects of subcomponents not demonstrated by the chemical procedures here utilized.     

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg²⁺ or Mn²⁺, and was free of interfering enzyme activities. Ca²⁺ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg²⁺ or Mn²⁺ was used as cofactor. In the presence of 5 millimolar Mg²⁺, Ca²⁺ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca²⁺. However, with Mn²⁺ (60 micromolar) as cofactor, Ca²⁺ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca²⁺ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg²⁺ or Mn²⁺ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg²⁺ as cofactor, but this effect was low with Mn²⁺. From these results we suggest that Ca²⁺ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.     

Specificity of sugar cane trehalase

An extract containing trehalase and invertase was prepared from apical internodes of sugar cane. The extract hydrolysed three glucosides: maltose, trehalose and sucrose. By reprecipitation with ammonium sulphate, maltase and trehalase activities appear to be due to different enzymes. As was also shown by differential inhibition and activation and by studies on the behaviour of both enzymes during growth, invertase and trehalase activities are attributed to different enzymes whose activities do not overlap. Invertase-free preparations confirm these results. Sucrose is a simple competitive inhibitor of sugar cane trehalase, excluding a regulatory role for this sugar. Sucrose was found at inhibitory levels in the first four apical internodes. A close correlation between sugar cane growth and invertase and trehalase levels was found in the apical internodes. Invertase has the greatest activity during growing, and trehalase reaches a maximum at maturity, prior to the flowering process. The high levels of trehalase in the flower suggest that the enzyme is involved in flowering or in related processes linked to seed formation.