Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment

The pathways of intracellular traffic involved in antigen processing and presentation have been defined by immunoelectron microscopy. The export pathway for class II histocompatibility molecules and the antigen import pathway meet in a peripheral endocytic compartment having all the molecular machinery believed to be required for antigen processing and presentation, including internalized surface immunoglobulins, proteolytic enzymes and invariant chains. This compartment defines a site where peptides from endocytosed antigen can bind class II molecules en route to the cell surface for presentation to T cells.     

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.     

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.     

Localization of the human GM-CSF receptor gene to the X-Y pseudoautosomal region

Mammalian sex chromosomes share a small terminal region of homologous DNA sequences, which pair and recombine during male meiosis. Alleles in this region can be exchanged between X and Y chromosomes and are therefore inherited as if autosomal. Genes from this so-called pseudoautosomal region (PAR) are present in two doses in both males and females, and escape inactivation of the X chromosome in females. Indirect evidence suggests that there must be several pseudoautosomal genes, and several candidates have been proposed. Until now, the only gene that has been unequivocally located in the PAR is MIC2, which encodes a cell-surface antigen of unknown function. We now report the localization of a gene of known function to this region--the gene for the receptor of the haemopoietic regulator, granulocyte-macrophage colony stimulating factor. The chromosomal localization of this gene may be important in understanding the generation of M2 acute myeloid leukaemia.     

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.     

Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.     

Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease

Amyloid B-protein/amyloid A4 is a peptide present in the neuritic plaques, neurofibrillary tangles and cerebrovascular deposits in patients with Alzheimer's disease and Down's syndrome (trisomy 21) and may be involved in the pathogenesis of Alzheimer's disease. Recent molecular genetic studies have indicated that amyloid protein is encoded as part of a larger protein by a gene on human chromosome 21 (refs 6-9). The amyloid protein precursor (APP) gene is expressed in brain and in several peripheral tissues, but the specific biochemical events leading to deposition of amyloid are not known. We have now screened complementary DNA libraries constructed from peripheral tissues to determine whether the messenger RNA encoding APP in these tissues is identical to that expressed in brain, and we identify a second APP mRNA that encodes an additional internal domain with a sequence characteristic of a Kunitz-type serine protease inhibitor. The alternative APP mRNA is present in both brain and peripheral tissues of normal individuals and those with Alzheimer's disease, but its pattern of expression differs from that of the previously reported APP mRNA.     

Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms

The conflict between the Mendelian theory of particulate inheritance and the observation of continuous variation for most traits in nature was resolved in the early 1900s by the concept that quantitative traits can result from segregation of multiple genes, modified by environmental effects. Although pioneering experiments showed that linkage could occasionally be detected to such quantitative trait loci (QTLs), accurate and systematic mapping of QTLs has not been possible because the inheritance of an entire genome could not be studied with genetic markers. The use of restriction fragment length polymorphisms (RFLPs) has made such investigations possible, at least in principle. Here, we report the first use of a complete RFLP linkage map to resolve quantitative traits into discrete Mendelian factors, in an interspecific back-cross of tomato. Applying new analytical methods, we mapped at least six QTLs controlling fruit mass, four QTLs for the concentration of soluble solids and five QTLs for fruit pH. This approach is broadly applicable to the genetic dissection of quantitative inheritance of physiological, morphological and behavioural traits in any higher plant or animal.     

Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin and lipocortin I

Significant future developments in the effective treatment of inflammatory diseases may arise from non-toxic dual inhibitors of both cyclooxygenase and lipoxygenase pathways in the arachidonate cascade. Inhibition of phospholipase A2(PLA2)(EC3.1.1.4), may provide such a dual action and recent research has concentrated on the role of PLA2-inhibitory proteins as possible anti-inflammatory agents. Blastokinin or uteroglobin is a steroid-induced rabbit secretory protein with PLA2-inhibitory activity. Its biochemical and biological properties have been extensively studied and its crystallographic structure has been resolved at 1.34 A (refs 15, 16). Lipocortins are a family of related proteins, which, it has been suggested, mediate the anti-inflammatory effects of glucocorticoids (for a review, see ref. 23). Some proteins of this group have been purified and the complementary DNA sequences of two human lipocortins are known. Lipocortins inhibit PLA2 in vitro, although their mechanism of action is still unclear. Recombinant lipocortin I inhibits eicosanoid synthesis in isolated perfused lungs from the guinea pig. Here, we report that synthetic oligopeptides corresponding to a region of high amino-acid sequence similarity between uteroglobin and lipocortin I have potent PLA2 inhibitory activity in vitro and striking anti-inflammatory effects in vivo.