A partial obstruction of 1 ureter was created in newborn rats and its effects were studied in the adult rat. The obstructed pelvis was found to be considerably enlarged. Nevertheless, the GFR (glomerular filtration rate) was only slightly decreased (10 per cent), completely compensated by increase on the contralateral, non-obstructed side. The reduction in GFR was less than the reduction in number of glomeruli (19 per cent), indicating a raised filtration rate per glomerulus. Water excretion was slightly increased and potassium excretion moderately decreased; sodium and osmolar excretion were not significantly affected. There was no correlation between these changes and the degree of pelvic enlargement. Thus, in this model, in which there is no urinary tract infection or pyelonephritis, partial obstructive uropathy caused less damage to the kidney function than might have been expected.
An Escherichia coli plasmid vector, pUN121, has been constructed which allows for positive selection of transformants harboring DNA inserts. The positive selection of transformants harboring DNA inserts. The vector is based on plasmid pTR262 (Roberts et al. Gene, 12, (1980), 123-127) in which the tetracycline resistance gene is under transcriptional control of the repressor protein coded by the phage lambda cI gene. This plasmid has been rearranged, using in vitro recombinant techniques including oligonucleotide mediated mutagenesis to yield a smaller plasmid (4.4 kb) with unique cloning sites for EcoRI, XmaI and SmaI in addition to the unique HindIII and BclI sites. The plasmid has a functional ampicillin resistance gene and the new restriction sites (EcoRI, XmaI and SmaI) when used for cloning, give rise to tetracycline resistant transformants.
Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.
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