IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.     

Sequence and specificity of a soluble lactose-binding lectin from Xenopus laevis skin.

A 16-kDa lactose-binding lectin comprises 5% or more of the soluble protein in Xenopus laevis skin. This lectin is mainly localized in the cytoplasm of granular gland cells. In response to stress, the lectin along with a variety of toxic and antibiotic peptides are released onto the skin surface by holocrine secretion. We have purified the lectin, sequenced tryptic peptides using tandem mass spectrometry and Edman degradation, and isolated full-length cDNA using a deduced oligonucleotide. Comparison of the cDNA and peptide sequences revealed expression of at least two isolectins, which differ in sequence at only two or three amino acids. Comparison of cDNA with complementary message by ribonuclease protection confirmed expression in approximately equal abundance of two nearly identical messages. The major soluble lactose-binding lectin expressed in Xenopus muscle is composed of these same isolectins, but at 100-fold lower levels. Similarities and distinctions in sequence and carbohydrate-binding specificity indicate that this lectin is a novel member of a family of soluble lactose-binding lectins expressed in a wide range of vertebrate tissues.     

Glycoprotein IV of bovine herpesvirus 1-expressing cell line complements and rescues a conditionally lethal viral mutant.

Glycoprotein IV (gIV) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus glycoprotein D, represents a major component of the viral envelope and a dominant immunogen. To analyze the functional role of gIV during BHV-1 replication, cell line BUIV3-7, which constitutively expresses gIV, was constructed and used for the isolation of gIV- BHV-1 mutant 80-221, in which the gIV gene was replaced by a lacZ expression cassette. On complementing gIV-expressing cells, the gIV- BHV-1 replicated normally but was unable to form plaques and infectious progeny on noncomplementing cells. Further analysis showed that gIV is essential for BHV-1 entry into target cells, whereas viral gene expression, DNA replication, and envelopment appear unchanged in both noncomplementing and complementing cells infected with phenotypically complemented gIV- BHV-1. The block in entry could be overcome by polyethylene glycol-induced membrane fusion. After passaging of gIV- BHV-1 on complementing cells, a rescued variant, BHV-1res, was isolated and shown to underexpress gIV in comparison with its wild-type parent. Comparison of the penetration kinetics of BHV-1 wild type, phenotypically complemented gIV- BHV-1, and BHV-1res indicated that penetration efficiency correlated with the amount of gIV present in virus particles. In conclusion, we show that gIV of BHV-1 is an essential component of the virion involved in virus entry and that the amount of gIV in the viral envelope modulates the penetration efficiency of the virus.     

Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo.

Recent reports on the possible contribution of the non-alpha-helical carboxy-terminal domain ("tail") of type III intermediate filament (IF) proteins to IF assembly have been controversial. To examine the importance and role of this domain, we have therefore engineered a Xenopus laevis vimentin cDNA to code for a tail-less polypeptide and have used it in combination with prokaryotic and eukaryotic expression systems. Here we show that tail-less vimentin, isolated from transfected bacteria (Escherichia coli), when used for assembly in vitro, forms normal-looking, loosely packed IFs. By viscometry we demonstrate that this tail-less vimentin assembles at an even higher rate and into longer IFs than wild-type vimentin. In vivo, i.e., by forced expression in transfected type III IF-free cultured epithelial cells, tail-less vimentin was also recovered in short fibrillar structures, in rodlets and in small as well as large spheroidal aggregates ("granules") that did not reveal any IF substructure. Surprisingly, however, spheroidal aggregate structures formed from the tail-deleted vimentin, were seen not only in the cytoplasm but also in the nucleus, indicating a role of the tail in higher order organization and compartmentalization of the vimentin IF system.     

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells

Summary A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.Correspondence to: T. Schweder     

Evidence supporting tight linkage of X-linked Emery-Dreifuss muscular dystrophy to the factor VIII:C gene.

In a large German family with Emery-Dreifuss muscular dystrophy (EDMD) linkage analysis was performed using the factor IX gene (F9), the factor VIII:C gene (F8), the anonymous DNA probe DXS52, and DXS15 as markers. Tight linkage was found between the EDMD locus and the F8 probe (Zmax = 1.19; theta max = 0.00), DXS15 (Zmax = 1.75; theta max = 0.00) and DXS52 (Zmax = 2.26; theta max = 0.00). Weak linkage was found to F9 (Zmax = 0.02; theta max = 0.43). The data from the literature and our results suggest that the gene locus of EDMD is close to F8 (confidence interval theta = 0-0.07). The new linkage data are useful for carrier detection and diagnosis of EDMD patients before onset of major clinical signs.     

Ion exchange and affinity chromatography in the scaleup of the purification of alpha-galactosidase from soybean seeds

Soybeans (Glycine max) contain an alpha-galactosidase that makes up a small fraction of the total protein of the seed. The properties of this enzyme are of interest because of its potential to convert the galactooligosaccharides, stachyose and raffinose, in soybean meal to sugars digestible in the human gastro intestinal tract and thereby increase potential uses of this vegetable protein source in human and animal foods. Study of this enzyme required the isolation of milligram quantities of electrophoretically pure protein from ground soybeans and therefore, scaleup of laboratory procedures by a factor of 300 times. Large scale acid precipitation, ammonium sulfate precipitation, and centrifugal recovery of the precipitated protein allowed alpha-galactosidase to be isolated from 45.5 kg soybean meal containing 17.1 kg protein, to obtain an enzyme extract with a specific activity of 90 to 100. A novel combination of strong anion exchange and cation exchange chromatography followed by Concanavalin-A affinity chromatography with a methyl alpha-D mannoside gradient gave alpha-galactosidase with an average specific activity of 56,000. Ion exchange chromatography preceding Concanavalin-A affinity chromatography allowed elimination of a relatively costly melibiose affinity chromatography step (which followed the Concanavalin-A column In the laboratory procedure) thereby making scaleup practical.