Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.     

Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase.

When chloramphenicol was added to a culture of Bacillus subtilis in early exponential growth, microscopic observation of cells stained by 4',6-diamidino-2-phenylindole showed nucleoids that had changed in appearance from irregular spheres and dumbbells to large, brightly stained spheres and ovals. In contrast, the addition of chloramphenicol to cultures in mid- and late exponential growth showed cells with elongated nucleoids whose frequency and length increased as the culture approached stationary phase. The kinetics of nucleoid elongation after the addition of chloramphenicol to exponential-phase cultures was complex. Immediately after treatment, the rate of nucleoid elongation was very rapid. The nucleoid then elongated steadily for about 4 min, after which the rate of elongation decreased considerably. Nucleoids of cells treated with 6-(p-hydroxyphenylazo)-uracil (an inhibitor of DNA synthesis) exhibited the immediate rapid elongation upon chloramphenicol treatment but not the subsequent changes. These observations suggest that axial filament formation during stationary phase (stage I of sporulation) in the absence of chloramphenicol results from changes in nucleoid structure that are initiated earlier, during exponential growth.     

A simple, sensitive and specific radioreceptor assay for beta-adrenergic antagonist drugs.

A radioreceptor assay for β-adrenergic blocking drugs described here is based on the ability of the blood content of drugs to compete with the binding of ³H-dihydroalprenolol (³H-DHA) to β-adrenergic receptors in calf cerebellar membranes. Plasma protein greatly inhibits the binding of ³H-DHA to β-receptors by binding the ³H-DHA so it is unavailable to the β-receptors. As little as 0.01 ml of human plasma in a final volume of 1 ml reduces binding 25–45%. Assays conducted on plasma dialysates can be performed without such inhibition. The radioreceptor assay is simple to perform as 100 samples can be processed in a morning. It is sensitive, detecting low nanomolar concentrations of plasma propranolol, and it is specific. No drugs clinically employed other than β-blocking agents compete for β-receptor binding. The assay detects all pharmacologically active metabolites of β-blocking drugs as well as the parent drug.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) is increased only 40% in these same preparations. The dissociation constant (K_D) of ¹²⁵I-HYP and the IC₅₀ of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC₅₀ of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of ¹²⁵I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (相似文献