Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum

Complexes of cell-surface receptors and their ligands are commonly internalized by endocytosis and enter a prelysosomal endosomal pathway for further processing. Fluorescence microscopy and video recording of living cells to trace the passage of ligand-receptor complexes has identified the endosomal compartment as an extensive network of tubular cisternae. Endocytosed material entering this reticulum enters discrete swellings, identified as multivesicular bodies by electron microscopy, which move along the reticulum towards the pericentriolar area.     

Small G proteins are expressed ubiquitously in lymphoid cells and do not correspond to Bcl-2

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth.

Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.     

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.     

A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by addition of telomeric repeat (TTAGGG)n

The instability of chromosomes with breaks induced by X-irradiation led to the proposal that the natural ends of chromosomes are capped by a specialized structure, the telomere. Telomeres prevent end-to-end fusions and exonucleolytic degradation, enable the end of the linear DNA molecule to replicate, and function in cell division. Human telomeric DNA comprises approximately 2-20 kilobases (kb) of the tandemly repeated sequence (TTAGGG)n oriented 5'----3' in towards the end of the chromosome, interspersed with variant repeats in the proximal region. Immediately subtelomeric lie families of unrelated repeat motifs (telomere-associated sequences) whose function, if any, is unknown. In lower eukaryotes the formation and maintenance of telomeres may be mediated enzymatically (by telomerase) or by recombination; in man the mechanisms are poorly understood, although telomerase has been identified in HeLa cells. Here we describe an alpha thalassaemia mutation associated with terminal truncation of the short arm of chromosome 16 (within band 16p13-3) to a site 50 kb distal to the alpha globin genes, and show that (TTAGGG)n has been added directly to the site of the break. The mutation is stably inherited, proving that telomeric DNA alone is sufficient to stabilize the broken chromosome end. This mechanism may occur in any genetic disease associated with chromosome truncation.     

(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL

The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone. It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates. As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.     

Localization of VP4 neutralization sites in rotavirus by three-dimensional cryo-electron microscopy

Three-dimensional structures of several spherical viruses have been determined by electron microscopy and X-ray crystallography. We report here the first three-dimensional structure of the complex between an intact virus and Fab fragments of a neutralizing monoclonal antibody. The antibody is against VP4, one of the two outer capsid proteins of rotaviruses. These large icosahedral viruses cause gastroenteritis in children and young animals and account for over a million human deaths annually. VP4 in these viruses has been implicated in several important functions such as cell penetration, haemagglutination, neutralization and virulence. Here we demonstrate that the surface spikes on rotavirus particles are made up of VP4. Antigenic sites are located near the distal ends of the spikes and two Fab fragments bind to each of the sixty spikes. The mass of the spike indicates that it is a dimer of VP4. The bilobed structure at the distal end of the spike may be involved in both the attachment to the cell and in viral penetration. A novel feature in the virus-Fab complex is the structural difference between the two chemically equivalent Fab fragments on each spike, which could be indicative of variations in the Fab elbow angles.