Murine antigen H-2.7: Phenotypic conversion of erythrocytes in radiation chimeras

By use of radiation chimeras produced between H-2.7⁺ and H-2.7^}- strains, A.SW and A.BY and B10.S(7R) and B10.S(9R), we demonstrate that the H-2.7 antigen can be passively attached to or detached from red blood cells. Thus, genetically H-2.7^}- red blood cells derived from H-2.7^}- bone marrow cells, gain H-2.7 antigen while maturing in the H-2.7⁺ host. Similarly, genetically H- 2.7⁺ red blood cells derived from H-2.7⁺ bone marrow cells become H-2.7^}- while maturing in H-2.7^– recipients. This behavior of the H-2.7 antigen is similar to that described for human Chido and Rodgers blood group antigens.Abbreviations used in this paper BMT bone marrow transfer - BSA bovine serum albumin - CT cytotoxicity test - HA hemagglutination - HBSS10 Hank's balanced salt solution containing 10% fetal calf serum - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

Revealing biases inherent in recombination protocols

Background  

The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.     

Effect of maurotoxin, a four disulfide-bridged toxin from the chactoid scorpion Scorpio maurus, on Shaker K+ channels.

Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.