A new way of stabilizing recombinant plasmids

A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp⁻. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained.     

Distribution of recombinant pro-urokinase in the rabbit blocked carotid artery

Distribution of recombinant pro-urokinase (PRU) in segments of blocked carotid arteries of rabbits was measured in 10, 20 and 30 minutes after i.v. administration of the RPU. Concentration of the latter in the bloodstream taken as 100% on the 3rd minute, after the infusion decreased to 42% in 10 min., 24% in 20 min., and 13% in 30 min. The RPU concentration decreased to 2% at the artery clamp point, to 20% at 10-15 cm from the clamp point, and remained constant for 10-30 min. A thrombolytic agent accumulated at the dead-end of the blocked artery, was not subject to rapid clearance in contrast to circulating pro-urokinase.     

A highly conserved interspecies V(H) in the human genome

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining V(H) of BSW17 with a human V(L) repertoire resulted in three light chains. The three V(L) chains were then combined with a human V(H) repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and V(H) complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 V(H) sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine V(H) sequence is associated with common antigen and epitope specificity.     

A new codon 31 (-C) mutant resulting in beta zero-thalassemia.

A new beta zero-thalassemia mutation, a frameshift mutation with deletion of a single cytosine nucleotide in codon 31, is described. The propositus, which is compound heterozygous for this mutation and the 17 beta A-T beta zero-thalassemia mutation, has the phenotype of severe beta-thalassemia major.     

Human extracellular recombinant phospholipase A2 induces an inflammatory response in rabbit joints

Phospholipase A2 (PLA2) enzymes hydrolyze membrane phospholipids liberating fatty acid and lysophospholipid. This event is thought to be the rate-limiting step in the generation of the lipid proinflammatory mediators, the eicosanoids and possibly platelet-activating factor. For this reason, extracellular forms of PLA2 have been postulated to be a component of the inflammatory cascade in certain biologic settings. In the synovial fluid of patients with inflammatory arthritides, substantial amounts of PLA2 activity have been found, and subsequently, one enzyme was purified, cloned, and expressed. Here we show that the pure recombinant enzyme free of any proinflammatory contaminants elicits a dramatic inflammatory, arthritogenic response when injected into the joint space of healthy rabbits. Within 24 h, extensive leukocyte infiltration and hyperplasia of the synovial lining cells were observed, and prostaglandin production in the joint space increased. In comparison, pancreatic PLA2(2) had little activity in this system, whereas a very inflammatory cobra venom enzyme was intermediate in its effects. In view of the presence of the enzyme in inflamed joints, the fact that its synthesis and secretion can be induced by proinflammatory cytokines and its proinflammatory biologic activity, we suggest that synovial PLA2 plays an exacerbating role in acute episodes in chronic inflammatory conditions such as rheumatoid arthritis.     

A new system for the expression of recombinant antibody in mammalian cells

Summary New type of plasmids named pEdHCG1 and pEdHC.were constructed. A combination of two plasmids containing the amplified gene product for the variable region of the antibody (Ab) heavy or light chains by the polymerase chain reaction (PCR) was co-transfected into COS cells to transiently express and in Chinese hamster ovary (CHO) cells to constitutively express chimeric Ab composed of the mouse-derived variable region and human-derived constant region.     

Effects of recombinant imperatoxin A (IpTxa) mutants on the rabbit ryanodine receptor

Imperatoxin A (IpTxa), a 3.7 kDa peptide from the African scorpion Pandinus imperator, is an agonist of the skeletal muscle ryanodine receptor (RyR1). In order to study the structure of the toxin and its effect on RyR1, IpTxa cDNA was PCR-amplified using 3 pairs of primers, and the toxin was expressed in E. coli. The toxin was further purified by chromatography, and various point mutants in which basic amino acids were substituted by alanine were prepared by site-directed mutagenesis. Studies of single channel properties by the planar lipid bilayer method showed that the recombinant IpTxa was identical to the synthetic IpTxa with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids (Lys19, Arg23, and Arg33) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when Lys8 was substituted with alanine. These results suggest that some basic amino acid residues in IpTxa are important for activation of RyR1, and that Lys8 plays an important role in regulating the gating mode of RyR1.     

A new method to measure post-traumatic joint contractures in the rabbit knee

A new device and method to measure rabbit knee joint angles are described. The method was used to measure rabbit knee joint angles in normal specimens and in knee joints with obvious contractures. The custom-designed and manufactured gripping device has two clamps. The femoral clamp sits on a pinion gear that is driven by a rack attached to a materials testing system. A 100 N load cell in series with the rack gives force feedback. The tibial clamp is attached to a rotatory potentiometer. The system allows the knee joint multiple degrees-of-freedom (DOF). There are two independent DOF (compression-distraction and internal-external rotation) and two coupled motions (medial-lateral translation coupled with varus-valgus rotation; anterior-posterior translation coupled with flexion-extension rotation). Knee joint extension-flexion motion is measured, which is a combination of the materials testing system displacement (converted to degrees of motion) and the potentiometer values (calibrated to degrees). Internal frictional forces were determined to be at maximum 2% of measured loading. Two separate experiments were performed to evaluate rabbit knees. First, normal right and left pairs of knees from four New Zealand White (NZW) rabbits were subjected to cyclic loading. An extension torque of 0.2 Nm was applied to each knee. The average change in knee joint extension from the first to the fifth cycle was 1.9 deg +/- 1.5 deg (mean +/- sd) with a total of 49 tests of these eight knees. The maximum extension of the four left knees (tested 23 times) was 14.6 deg +/- 7.1 deg, and of the four right knees (tested 26 times) was 12.0 deg +/- 10.9 deg. There was no significant difference in the maximum extension between normal left and right knees. In the second experiment, nine skeletally mature NZW rabbits had stable fractures of the femoral condyles of the right knee that were immobilized for five, six or 10 weeks. The left knee served as an unoperated control. Loss of knee joint extension (flexion contracture) was demonstrated for the experimental knees using the new methodology where the maximum extension was 35 deg +/- 9 deg, compared to the unoperated knee maximum extension of 11 deg +/- 7 deg, 10 or 12 weeks after the immobilization was discontinued. The custom gripping device coupled to a materials testing machine will serve as a measurement test for future studies characterizing a rabbit knee model of post-traumatic joint contractures.     

Cloning and expression of recombinant rabbit fertilin

Fertilin is a sperm surface protein complex which is reported to play an essential role in sperm-egg fusion in mammals. It is comprised of two related subunits, α and β, both of which are glycosylated and have cytoplasmic and extracellular domains. This protein has been reported to play an essential role in sperm-egg fusion in mammals. We report on the cloning and sequencing of the complete cDNA sequences of both subunits from rabbit testis, and the production of recombinant proteins for testing their potential as antigens for use in an immunocontraceptive vaccine to control wild rabbit populations. The cDNAs for rabbit fertilin α and β (Genbank accession numbers, U46069 and U46070) are predicted to encode proteins of 9l9 and 751 amino acids, respectively, and to show significant levels of homology to fertilin subunits isolated from other species. Analysis of the predicted protein sequences of fertilin α but not β reveals the presence of 21 direct repeats of the hexameric sequence A/PPPPEA at the extreme carboxy terminus, similar to what has been described for a fertilin α gene isoform in the monkey. DNA sequences corresponding to the predicted mature α and β fertilin subunits were individually cloned into a bacterial expression system, and the recombinant proteins were used to raise polyclonal antibodies in mice. These antibodies detect components of the native fertilin complex from rabbit sperm. © 1996 Wiley-Liss, Inc.     

Sequences of four new members of the V H7183 gene family in BALB/c mice

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U04227-U04232