Mathematical modeling for bioprocess optimization of a protein drug, uricase, production by Aspergillus welwitschiae strain 1-4.

Microbial uricase is effective protein drug used to treat hyperuricemia and its complications, including chronic gout, also in prophylaxis and treatment of tumor lysis and organ transplants hyperuricemia. Uricase is commonly used as diagnostic reagent in clinical analysis for quantification of uric acid in blood and other biological fluids. Also, it can be used as an additive in formulations of hair coloring agents. A newly isolated strain, Aspergillus sp. 1-4, was able to produce extracellular uricase on a medium containing uric acid as inducer. Phylogenetic analysis based on ITS region sequence analysis and phenotypic characteristics showed that Aspergillus sp. strain 1-4 is closely related to Aspergillus welwitschiae and its nucleotide sequence was deposited in the GenBank database and assigned sequence accession number MG323529. Statistical screening using Plackett-Burman design with 20 runs was applied to screen fifteen factors for their significance on uricase production by Aspergillus welwitschiae. Results of statistical analysis indicated that incubation time has the most significant positive effect on uricase production followed by yeast extract and inoculum size with the highest effect values of 13.48, 5.26 and 4.75; respectively. The interaction effects and optimal levels of these factors were evaluated using central composite design. The maximum uricase production was achieved at incubation time (5 days), yeast extract (2 g/L) and inoculum size (4 mL/50 mL medium) are the optimum levels for maximum uricase production (60.03 U/mL). After optimization, uricase production increased by 3.02-folds as compared with that obtained from the unoptimized medium (19.87 U/mL).

Active uptake and extravesicular storage of m-iodobenzylguanidine in human neuroblastoma SK-N-SH cells.

Radio-iodinated m-iodobenzylguanidine (MIBG), an analogue of the neurotransmitter norepinephrine (NE), is increasingly used in the diagnosis and treatment of neural crest tumors. Active uptake and subsequent retention of MIBG and NE was studied in human neuroblastoma SK-N-SH cells. Neuron-specific uptake of [125I]MIBG and [3H]NE saturated at extracellular concentration of 10(-6) M and exceeded by 20-30-fold that by passive diffusion alone. A minimum of 50% of accumulated MIBG remained permanently stored but the SK-N-SH cells were incapable of retaining recaptured [3H]NE. [125I]MIBG was displaced from intracellular binding sites by unlabeled MIBG with 10-fold higher potency than by unlabeled NE. MIBG stored in SK-N-SH cells was insensitive to depletion by the inhibitor of granular uptake reserpine (RSP) and was not precipitated in a granular fraction by differential centrifugation. Only few electron-dense granules were found in these cells by electron microscopy. In contrast, MIBG storage in PC-12 pheochromocytoma cells which contained many storage granules, was sensitive to RSP and part of accumulated drug was recovered in a granular fraction. Accordingly, storage of MIBG in the SK-N-SH neuroblastoma cells is predominantly extravesicular and thus essentially different from that of biogenic amines in normal adrenomedullary tissue or in pheochromocytoma tumors, while sharing with these tissues a common mechanism of active uptake.

Potentiation of glucocorticoid-induced lysis in refractory and resistant leukemia cells by inhibitors of ADP-ribosylation.

Meta-iodo-benzylguanidine (MIBG; 3 x 10(-5) M), a novel inhibitor of mono(ADP-ribosylation)-and the general ribosylation inhibitor nicotinamide (NA; 5-20 mM) both stimulated the glucocorticoid-mediated lysis of sensitive L1210 leukemia cells and even induced susceptibility in various human and murine lines refractory or resistant to dexamethasone (DEX). Potentiation and induction of DEX-sensitivity by ADP-ribosylation inhibitors was accompanied by an increase in saturable 3H-DEX binding sites and by a 2-3 fold increase in the affinity of intracellular receptors for hormone binding. Moreover, the ribosylation inhibitors converted the glucocorticoid antagonist RU-486 into a potent agonist for cytolysis of L1210 cells. We conclude that the cytolytic action of glucocorticoid hormones in leukemic cells is negatively controlled by (mono)ADP-ribosylation of receptor proteins.