Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli

The bacterial membrane serves both as a cell organelle and as a barrier for segregating the metabolically active cytoplasm from the extracellular milieu. Thus we can use plasmid vectors designed to produce a hybrid protein containing an efficient signal peptide coupled to the amino terminus of the cloned heterologous protein (secretion cloning vectors) for the production of proteins which are insoluble, proteolytically sensitive, or bacteriocidal when produced in the cytoplasm of Escherichia coli. We demonstrate that human granulocyte-macrophage colony stimulating factor can be isolated as an active species only after transport into the bacterial periplasm. Production of the protein in the bacterial cytoplasm is bacteriocidal. We also demonstrate that biologically active human interleukin 4 appears only after transport of the protein into the bacterial growth medium. The protein forms membrane-associated aggregates in the cytoplasm, and demonstrates an active but nonnative conformation when expressed in the periplasm. This may correlate with the affinity of the interleukin 4 molecule for negatively charged macromolecules, including bacterial membrane components and bacterial lipopolysaccharides, which may alter the folding pathway inside the cell.     

RESPONSE OF ANTARCTIC FUR SEALS TO IMMOBILIZATION WITH KETAMINE, A KETAMINE-DIAZEPAM OR KETAMINE-XYLAZINE MIXTURE, AND ZOLETIL®

Adult Antarctic fur seals (Arctocephalus gazella) were immobilized with Zoletil^® ( n = 172), ketamine ( n = 30), ketamine mixed with diazepam ( n = 23) and with ketamine mixed with xylazine ( n = 45). Response to all drugs was highly variable. There was a relationship between dose rate and level of immobilization in females given Zoletil^®. Males were slightly more sensitive to Zoletil^® than females but this could have been due to the greater body mass and lower mass-specific metabolic rate of males. The dose required to achieve a level of immobilization declined with greater body mass for Zoletil^® and ketamine but not for ketamine-diatepam. Ketamine and ketamine-sedative mixtures commonly caused mild tremoring and occasionally caused convulsions. Neither reaction was seen with Zoletil^®. Mean doses were, Zoletil^® 1.5 mg/ kg, ketamine 6.9 mg/kg, ketamine-diazepam 6.3 mg/kg ketamine and 6.3 μg/kg diazepam, and ketamine-xylazine 7.3 mg/kg ketamine and 0.62 mg/ kg xylazine. Zoletil^® performs at least as well on Antarctic fur seals as ketamine but it may cause respiratory depression. The dose of ketamine required for Antarctic fur seals was greater than for most other species of seals.     

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.     

Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli

Thioredoxin was cross-linked to a membrane fraction in vivo using the heterobifunctional photoreactive cross-linking reagent p-azidophenacyl bromide, chosen to couple thioredoxin via its highly reactive thiol. Under mild reaction conditions, a significant amount of thioredoxin (30%) was rapidly cross-linked to the crude membrane fraction. The cross-linking reaction was selective, with thioredoxin purified 15-fold in the cross-linked membrane fraction. Membrane fractionation studies showed that thioredoxin associated with the inner membrane and with a hybrid membrane fraction. This hybrid membrane fraction banded at a density between the inner and outer membranes. This result is consistent with the localization of thioredoxin in association with the bacterial membrane adhesion sites first described by Bayer (Bayer, M. (1968) J. Gen. Microbiol. 53, 395-404). Association of thioredoxin with the membrane adhesion sites defines a structure corresponding to the osmotically sensitive cytoplasmic compartment (Lunn, C. A., and Pigiet, V. (1982) J. Biol. Chem. 257, 11424-11430).     

Decontamination of aqueous solutions of biological stains.

Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic.     

Macrophage dependence of peripheral sensory nerve regeneration: possible involvement of nerve growth factor.

The levels of NGF and NGF receptor mRNA, the degree of macrophage recruitment, and the ability of sensory and motor axons to regenerate were measured in C57BL/Ola mice, in which Wallerian degeneration following a nerve lesion is very slow. Results were compared with those from C57BL/6J and BALB/c mice, in which degeneration is normal. We found that in C57BL/Ola mice, apart from the actual lesion site, recruitment of macrophages was much lower, levels of mRNA for both NGF and its receptor were raised only slightly above normal, and sensory axon regeneration was much impaired. Motor axons regenerated quite well. These results provide in vivo evidence that macrophage recruitment is an important component of NGF synthesis and of sensory (but not motor) axon maintenance and regrowth.