Construction of an Escherichia coli export-affinity vector for expression and purification of foreign proteins by fusion to cyclomaltodextrin glucanotransferase.

A novel export-affinity fusion vector employing the gene encoding cyclomaltodextrin glucanotransferase (CGTase; cgt) from Bacillus circulans var. alkalophilus (ATCC 21783) is described. CGTase binds to various sugar polymers, which makes it simple to purify it to near homogeneity in a single step. The CGTase fusion protein vector was constructed by deleting the translational stop codons from the gene encoding CGTase (cgt) by in vitro mutagenesis. As models, genes encoding Escherichia coli alkaline phosphatase (APase; phoA) and Bacillus stearothermophilus (ATCC 12980) alpha-amylase (BStA; amy) were fused to cgt. Overexpression of wild type CGTase and the hybrid proteins under the control of the lac promoter caused a 'leaky phenotype' in E. coli, the outer membrane became permeable, which enabled the adsorption of the fusion proteins directly from the culture medium onto alpha-cyclodextrin (alpha-CD) coupled agarose. The hybrid proteins were eluted from the column with alpha-CD solution under mild conditions at pH 7.5. The CGTase-APase' fusion had a good in vivo stability, whereas the CGTase-BStA' was less stable. In the latter case, according to protein sequencing, the proteolytically sensitive site was on the BStA' side of the fusion. The C-terminus of CGTase was stable against proteolysis as shown by narrow pH range isoelectric focusing. The fused enzymes retained their biological activities.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic beta-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba2+ was not inhibited by D-glucose. Ba2+ served as a substitute for Ca2+ both in maintaining the glucose metabolism after removal of extracellular Ca2+ and making it possible for glucose to stimulate insulin release. Furthermore, Ba2+ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the beta-cell content of cyclic AMP. It is likely that the observed actions of Ba2+ are mediated by Ca2+, since Ca2+ -dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba2+ specifically.     

Glutaraldehyde Inactivation of Virus in Tissue

High concentrations of influenza virus and T3 coliphage were inoculated into mouse tissue blocks. Exposure of the inoculated tissue blocks to 5% alkaline glutaraldehyde resulted in rapid inactivation of both viral agents.     

Novel post-translational modifications of Smad2 identified by mass spectrometry

Smad2 is a crucial component of transforming growth factor-β (TGFβ) signaling, and is involved in the regulation of cell proliferation, death and differentiation. Phosphorylation, ubiquitylation and acetylation of Smad2 have been found to regulate its activity. We used mass spectrometry to search for novel post-translational modifications (PTMs) of Smad2. Peptide mass fingerprinting (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, phosphorylated and palmitoylated. Sequencing of selected peptides validated methylation at Gly122 and hydroxylation at Trp18 of Smad2. We also observed a novel, so far unidentified modification at Tyr128 and Tyr151. Our observations open for further exploration of biological importance of the detected PTMs. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

A rapidly evolving region in the immunoglobulin heavy chain loci of rat and mouse: postulated role of (dC-dA)n.(dG-dT)n sequences

The nucleotide sequences of the introns that are located between the C4 exon and the first membrane exon of mouse and rat immunoglobulin epsilon-chain genes have been determined. The rat intron sequence was found to contain four separate clusters of repetitive sequences all of which consisted of (dC-dA)n.(dG-dT)n dinucleotide repeats. A comparison between this chromosomal region in mouse and rat revealed four deletions or duplications, three of which have occurred inside or at the borders of the CA clusters. Rearrangements have occurred inside or at the borders of all four repeats after the evolutionary separation of mouse and rat. The sequence comparison reveals in addition a duplication, connected to the CA repeats, which has occurred early in evolution, before the evolutionary divergence of mouse and rat. These findings suggest that (dC-dA)n.(dG-dT)n sequences are potential targets for recombination events.     

The islets of Langerhans in ducks and chickens with special reference to the argyrophil reaction

Summary The pancreas of birds is a suitable object for studying the A and B cells separately, since the two cell systems are topografically almost entirely segregated in the form of light (= B cells) and dark (= A cells) islets of Langerhans.On the whole in the chicken and duck the actual distribution of the light islets into different size classes followed the same regular pattern previously found in the rat and man. In the body of the pancreas, containing the great majority of islets, the volume distribution curves thus appeared symmetrical.With the silver impregnation method used a distinct argyrophil reaction in both types of islets was obtained on paraffin sections of the pancreas. According to the presence or absence of blackening, the cells of the dark islets could be divided into two distinct fractions. Especially in the duck the silver-positive cells were grouped in a characteristic way along the walls of the capillaries. Ducks and chickens are not the only animals in which it is possible to identify an argyrophil fraction in what the usual granule stains had shown to be A cells. Parallel studies of various mammals are in complete agreement with these observations. It is, however, still uncertain whether we are here dealing with differences in function, age etc. in one and the same type of cell or with two completely different kinds. No correlation between the argyrophil reaction in the dark islet cells and their content of SH and SS groups or tryptophane could be established.This work was aided by grants from the Swedish Medical Research Council.