Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid

Degradation of a synthetic oligodeoxynucleoside phosphorothioate was studied in six systems used for antisense inhibition experiments. Oligodeoxynucleoside phosphorothioates were degraded very slowly at 37 degrees C in all of the systems studied. Measured half-lives of pentadecamers were 12 +/- 1 h in rabbit reticulocyte lysate, 7 +/- 1 h in HeLa cell postmitochondrial extract, 14 +/- 2 h in RPMI 1640 with 10% fetal bovine serum, 8 +/- 1 h in undiluted fetal bovine serum, 9 +/- 1 h in adult human serum, and 19 +/- 7 h in rat cerebrospinal fluid.     

Oligodeoxynucleotide stability in subcellular extracts and culture media

Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences. Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum.     

Lyophilization of rumen fluid for use in culture media

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

alpha-Oligodeoxynucleotide stability in serum, subcellular extracts and culture media

Degradation of a synthetic alpha-oligodeoxynucleotide was studied in order to compare its survival with naturally occurring beta-oligodeoxynucleotides in five systems used for antisense hybridization arrest experiments. In contrast to beta-oligodeoxynucleotides, alpha-oligodeoxynucleotides were not detectably degraded over 24 h at 37 degrees C in HeLa cell postmitochondrial cytoplasmic extract or RPMI 1640 with 10% fetal bovine serum, and showed significant survival after 24 h at 37 degrees C in rabbit reticulocyte lysate, fetal bovine serum and human serum.     

Enzymatic release of mycobacteria in natural media]

Polysaccharases release mycobacteria from natural environment. The enzymatic activity works both on the microbial adherence polysaccharides and on the support surfaces (cellulose). The release of mycobacteria from natural environment increases both the number of isolates and the number of species of mycobacteria.     

Express method of determining phenylacetic acid in the culture broth during the biosynthesis of benzylpenicillin

Phenylacetate acid (PAA) is transferred by extraction from the fermentation broth filtrate into toluol. The extract is applied to a Silufol plate with an aluminium foil lining (silica gel sorbent, Czechoslovakia). Reference solutions of PAA are also applied to the same plate. The reference and test solutions are applied dropwise (spots of 5--6 x 10(-3)m in diameter). For PAA development the spots are sprayed with a freshly prepared saturated solution of potassium manganate in 6N H2SO4. PAA of the test samples is developed as a dull ring against grey background and that of the reference solution is developed as a circle. The amount of PAA in the spot is determined by using correlation between the spot area and the amount of PAA applied. One plate of 225 X 10(-4) m2 can be used for about 300 analyses. One analysis takes 300--600 seconds.     

Lyophilization of rumen fluid for use in culture media.

The supernatant from centrifugation at 1,000 x g of strained rumen fluid was lyophilized, and the residue and sublimate fractions were used to replace fresh rumen fluid in a complete roll tube medium for enumeration of total rumen bacteria. Most of the growth-supporting nutrients in fresh rumen fluid were found in the residue fraction. With one exception, no significant differences were found in total bacterial numbers either by roll tube or most-probable-number procedures when lyophilized rumen fluid residue was substituted for fresh rumen fluid. Lyophilized rumen fluid residue was stable for at least 5 months at room temperature. Rumen fluid supernatant from centrifugation at 1,000 x g had a mean density of 1.005 +/- 0.03 g/ml and contained 1.56% +/- 0.30% dry matter. On the basis of these values, 15.68 mg of lyophilized rumen fluid residue is equivalent to 1 ml of rumen fluid supernatant from centrifugation at 1,000 x g.     

A comparative evaluation of the pharmacokinetics of different forms of benzathine benzylpenicillin]

Comparative randomized opened pharmacokinetic evaluation of benzathine benzylpenicillin in three dosage forms was performed. Benzathine benzylpenicillin was used as extencilline (2.4 million U or 1.2 million U, "Rh?ne-Poulenc Rorer", France) and as bicillin-5 (1.5 million U, "Synthesis" Russia). 33 patients were included in investigation (23 women and 10 men aged 16-60 years). 25 persons had verified rheumatism without blood circulation failure signs, 4--had chronic tonsillitis and 4 were healthy volunteers. Benzylpenicillin concentration was estimated by microbiology test in blood samples taken at 1, 3, 24 hours and 7, 14 and 21 days after intramuscular drug injection. After 2.4 million U extencilline injection (12 patients) its concentration, was at the inhibition level for beta-hemolytic streptococcus group A (25 ng/ml) for 3 weeks-period in 83.3 per cent of patients. After 1.2 million U extencilline injection (10 patients) or 1.5 million U bicillin-5 injection (12 patients) the above mentioned concentration was achieved on the 21st day in 30 and 0 per cent of patients respectively. Thus the treatment with benzathine benzylpenicillin at the 1.2 million U dose in the form of extencilline or bicillin-5 doesn't provide adequate antistreptococcal concentration in blood in prolonged period and is not suitable for correct rheumatism prophylaxis in adult patients.     

The effect of salt concentration on auxin stability in culture media

The concentrations of indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were followed for 35 days in cell-free liquid medium containing 100, 50, or 0% Murashige-Skoog (MS) salt base. Although the concentrations of NAA or 2,4-D remained constant the level of IAA decreased to only 11% of the original concentration after 35 days in the presence of 100% MS salt base. The observed rate of IAA degradation was accelerated by the presence of MS salts.     

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were -modified Eagle's minimum essential medium (MEM-) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.     

Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha‐ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2‐methyl‐1,3‐thiazolidine‐2,4‐dicarboxylic acid and 2‐(2‐carboxyethyl)?1,3‐thiazolidine‐2,4‐dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha‐ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l ‐methionine (met) and l ‐tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed‐batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti‐oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant‐free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759–770, 2017