The bcl-2 gene encodes a novel G protein

Little is known about the biochemical or functional nature of the proteins encoded by the bcl-2 gene, which undergoes chromosomal translocation in approximately 85% of follicular lymphoma, 20% of diffuse large cell lymphoma and 10% of chronic lymphocytic leukaemia of B cells. Translocation of bcl-2 sequences from chromosome 18 to the JH segment of the immunoglobulin gene at chromosome band 14q32 in B cells results in deregulated expression of this gene, causing high steady state levels of bcl-2 messenger RNA2. DNA sequence data indicate that bcl-2 encodes two proteins by virtue of alternative splicing, designated as Bcl-2 alpha and Bcl-2 beta, with relative molecular masses of 26,000 and 22,000 respectively. Cell fractionation experiments indicate that the bcl-2 alpha gene product is located at the inner surface of the cell membrane, suggesting a possible role in mitogenic signal transduction. We report here that Bcl-2 alpha has GTP-binding activity and a protein sequence that suggests it belongs to the small molecular weight GTP-binding protein (G protein) family.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

Arginine vasopressin enhances pHi regulation in the presence of HCO3- by stimulating three acid-base transport systems

Growth factors raise intracellular pH (pHi) by stimulating Na+/H+ exchange in the absence of HCO3-. In mutant cells that lack the Na+/H+ exchange activity, this alkalinization does not occur, and the cells do not proliferate without artificial elevation of pHi. It has therefore been widely suggested that an early pHi increase is a necessary signal for mitogenesis. In the presence of HCO3- however, growth factors fail to raise pHi in A431 cells, renal mesangial cells and 3T3 fibroblasts. In mesangial cells, arginine vasopressin (AVP) raises pHi in the absence of HCO3-, but lowers it when HCO3- is present; growth is stimulated under both conditions. We report here that, in the presence of HCO3-, AVP stimulates two potent HCO3- transporters, as well as the Na+/H+ exchanger. These are the Na+-dependent and Na+-independent Cl-/HCO3- exchangers. Our results indicate that AVP causes acidification in the presence of HCO3- because, at the resting pHi, it stimulates Na+-independent Cl-/HCO3- exchange (which lowers pHi) more than it stimulates the sum of Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange (both of which raise pHi). The stimulation of three acid-base transporters by the growth factor AVP greatly enhances the ability of the cell to regulate pHi.     

Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum

The primary event in the pathogenesis of severe malaria in Plasmodium falciparum infection is thought to be adherence of trophozoite- and schizont-infected erythrocytes to capillary endothelium, a process called sequestration. Identifying the endothelial molecules used as receptors is an essential step in understanding this disease process. Recent work implicates the membrane glycoprotein CD36 (platelet glycoprotein IV; refs 2-5) and the multi-functional glycoprotein thrombospondin as receptors. Although CD36 has a widespread distribution on microvascular endothelium, it may not be expressed on all capillary beds where sequestration occurs, especially in the brain. The role of thrombospondin in cell adhesion, in vitro or in vivo, is less certain. We have noticed that some parasites bind to human umbilical-vein endothelial cells independently of CD36 or thrombospondin. To screen for alternative receptors, we have developed a novel cell-adhesion assay using transfected COS cells, which confirms that CD36 is a cell-adhesion receptor. In addition, we find that an endothelial-binding line of P. falciparum binds to COS cells transfected with a complementary DNA encoding intercellular adhesion molecule-1. As this molecule is widely distributed on capillaries and is inducible, this finding may be relevant to the pathogenesis of severe malaria.     

The Drosophila posterior-group gene nanos functions by repressing hunchback activity

The development of the body plan in the Drosophila embryo depends on the activity of maternal determinants localized at the anterior and posterior of the egg. These activities define both the polarity of the anterior-posterior (AP) axis and the spatial domains of expression of the zygotic gap genes, which in turn control the subsequent steps in segmentation. The nature and mode of action of one anterior determinant, the bicoid(bcd) gene product, has recently been defined, but the posterior determinants are less well characterized. At least seven maternally acting genes are required for posterior development. Mutations in these maternal posterior-group genes result in embryos lacking all abdominal segments. Cytoplasmic transplantation studies indicate that the maternally encoded product of the nanos(nos) gene may act as an abdominal determinant, whereas the other maternal posterior-group genes appear to be required for the appropriate localization and stabilization of this signal. Here we show that the lack of the nos gene product can be compensated for by eliminating the maternal activity of the gap gene hunchback (hb). Embryos lacking both of these maternally derived gene products are viable and can survive as fertile adults. These results suggest that the nos gene product functions by repressing the activity of the maternal hb products in the posterior of the egg.     

Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement

Oligodendrocytes synthesize myelin in the central nervous system and maintain it in lamellar sheaths around axons. Techniques for studying oligodendrocyte development in vitro can be used, indirectly, to investigate the myelin injury that occurs in human and experimental demyelinating disease. Cell-mediated immune mechanisms are necessary but not sufficient to induce myelin damage in vivo; more recently complement has also been implicated in the pathogenesis both of multiple sclerosis and experimental allergic encephalomyelitis. Previously we have demonstrated that antibody-independent complement activation occurs in vitro at the oligodendrocyte surface. Here we show that the ensuing oligodendrocyte injury is reversible, and that recovery involves the release of membrane-attack complex-enriched vesicles from the surface of viable cells. The demonstration of morphologically and immunochemically identical vesicles in the cerebrospinal fluid of patients with multiple sclerosis suggests that reversible complement-mediated injury contributes to myelin damage in vivo.     

A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin

Antibodies and growth factors have been chemically coupled to different toxins to produce cytotoxic molecules that selectively kill cells bearing appropriate antigens or receptors. Antibody-toxin conjugates (immunotoxins) produced using conventional chemical coupling techniques have several undesirable characteristics. The smallest binding unit of an antibody is an Fv fragment which consists of a light and heavy chain variable domain. Recently, active single chain Fv fragments of antibodies have been produced in Escherichia coli by attaching the light and heavy chain variable domains together with a peptide linker. Here we describe the construction and expression in E. coli of a single chain antibody toxin fusion protein, anti-Tac(Fv)-PE40, in which the variable regions of anti-Tac, a monoclonal antibody to the p55 subunit of the human interleukin-2 receptor, are joined in peptide linkage to PE40, a modified form of Pseudomonas exotoxin lacking its binding domain. Anti-Tac(Fv)-PE40 was very cytotoxic to two interleukin-2 receptor-bearing human cell lines but was not cytotoxic to receptor-negative cells.