Development of recombinant human granulocyte colony-stimulating factor (nartograstim) production process in Escherichia coli compatible with industrial scale and with no antibiotics in the culture medium.

The granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that has important clinical applications for treating neutropenia. Nartograstim is a recombinant variant of human G-CSF. Nartograstim has been produced in Escherichia coli as inclusion bodies (IB) and presents higher stability and biological activity than the wild type of human G-CSF because of its mutations. We developed a production process of nartograstim in a 10-L bioreactor using auto-induction or chemically defined medium. After cell lysis, centrifugation, IB washing, and IB solubilization, the following three refolding methods were evaluated: diafiltration, dialysis, and direct dilution in two refolding buffers. Western blot and SDS-PAGE confirmed the identity of 18.8-kDa bands as nartograstim in both cultures. The auto-induction medium produced 1.17 g/L and chemically defined medium produced 0.95 g/L. The dilution method yielded the highest percentage of refolding (99%). After refolding, many contaminant proteins precipitated during pH adjustment to 5.2, increasing purity from 50 to 78%. After applying the supernatant to cation exchange chromatography (CEC), nartograstim recovery was low and the purity was 87%. However, when the refolding solution was applied to anion exchange chromatography followed by CEC, 91%-98% purity and 2.2% recovery were obtained. The purification process described in this work can be used to obtain nartograstim with high purity, structural integrity, and the expected biological activity. KEY POINTS: • Few papers report the final recovery of the purification process from inclusion bodies. • The process developed led to high purity and reasonable recovery compared to literature. • Nartograstim biological activity was demonstrated in mice using a neutropenia model.

The role of angiotensin-converting enzyme inhibitors in reducing ventricular remodeling after myocardial infarction.

Ventricular remodeling is a pathologic change in the size and shape of the heart after myocardial infarction. Human and animal studies have described the mechanisms responsible for the thinning and enlargement that progresses for years beyond the initial infarction. As a result of elevations in preload and afterload, ventricular pressures increase, and changes occur in both the infarcted and uninfarcted regions of the ventricle, increasing overall heart size. Recent investigation has demonstrated that the initiation of angiotensin-converting enzyme (ACE) inhibitor drugs after myocardial infarction reduces both systolic and diastolic wall stresses, thereby averting changes in heart size. These findings are significant, as increases in heart size and ventricular volumes have proved to be powerful predictors of early mortality after myocardial infarction.

Ventricular remodeling following myocardial infarction: a description of the pathologic process, current investigation, and suggested therapy.

The pathophysiologic process of ventricular remodeling after AMI involves an alteration in myocardial cell contraction. The stretching and redistribution of myocardial cells in the ischemic area promote dyssynergic contraction and an overall reduction in ventricular function. Expansion of the infarcted area and volume-overload hypertrophy of the uninfarcted area remodel the shape of the ventricle. Ventricular enlargement and dilation are associated with early mortality and morbidity. This has prompted further study to identify measures that can attenuate the process. Limited investigation on human subjects suggests that ACE inhibition reduces ventricular wall stress and preserves ventricular shape and function. A multicenter trial, SAVE, is under way to study the effects of long-term captopril therapy for patients suffering from AMI. This study and future investigations will focus on inhibition of ventricular remodeling following AMI in the hope of reducing symptomatic CHF and mortality.

Diamine Oxidase Activity in Different Physiological Stages of Helianthus tuberosus Tuber.

Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Delta(1)-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the K(m) of the enzyme for putrescine was of the order of 10(-4) molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation.