Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.
Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand. (+info)
Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles.
A messenger ribonucleoprotein (mRNP) particle containing the mRNA coding for the myosin heavy chain (MHC mRNA) has been isolated from the postpolysomal fraction of homogenates of 14-day-old chick embryonic muscles. The mRNP sediments in sucrose gradient as 120 S and has a characteristic buoyant density of 1.415 g/cm3, which corresponds to an RNA:protein ratio of 1:3.8. The RNA isolated from the 120 S particle behaved like authentic MHC mRNA purified from chick embryonic muscles with respect to electrophoretic mobility and ability to program the synthesis of myosin heavy chain in a rabbit reticulocyte lysate system as judged by multi-step co-purification of the in vitro products with chick embryonic leg muscle myosin added as carrier. The RNA obtained from the 120 S particle was as effective as purified MHC mRNA in stimulating the synthesis of the complete myosin heavy chains in rabbit reticulocyte lysate under conditions where non-muscle mRNAs had no such effect. Analysis of the protein moieties of the 120 S particle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows the presence of seven distinct polypeptides with apparent molecular weights of 44,000, 49,000, 53,000, 81,000, 83,000, and 98,000, whereas typical ribosomal proteins are absent. These results indicate that the 120 S particles are distinct cellular entities unrelated to ribosomes or initiation complexes. The presence of muscle-specific mRNAs as cytoplasmic mRNPs suggests that these particles may be involved in translational control during myogenesis in embryonic muscles. (+info)
Characterization of nuclear structures containing superhelical DNA.
Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated. (+info)
The nucleoprotein of Marburg virus is target for multiple cellular kinases.
The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404-432; II*, amino acids 446-472; III*, amino acids 484-511; IV*, amino acids 534-543; V*, amino acid 549; VI*, amino acids 599-604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*-V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis. (+info)
Model studies of chromatin structure based on X-ray diffraction data.
Model calculations are presented in order to interpret the X-ray diffraction diagrams given by chromatin gels. It is shown that by taking into account the hydration of chromatin subunits, the problem of calculating the interference function in concentrated gels is greatly simplified. In this way it is spossible to fully interpret the influence of concentration on the position and intensity of the various rings present in the X-ray diffraction patterns. The possibilities and limitations of models based on spherical symmetry are also discussed. It is concluded that each chromatin subunit most likely contains three turns of DNA in each 200 base pairs segment surrounding a central protein core. With the method presented here it is possible to test if other models of chromatin based on different kinds of evidence are compatible with the X-ray diffraction data. (+info)
Non-purulent meningoencephalomyelitis of a Pacific striped dolphin (Lagenorhynchus obliquidens). The first evidence of morbillivirus infection in a dolphin at the Pacific Ocean around Japan.
On March 22, 1998, a mature, male, hyposthenic Pacific striped dolphin (Lagenorhynchus obliquidens) was stranded at Aoshima Beach in Miyazaki prefecture, Japan. A necropsy performed 14 hr after death revealed mild diffuse congestion and edema of the leptomeninges and mild pulmonary atelectasis. Histopathologically, non-purulent inflammatory were observed throughout the cerebrum, thalamus, midbrain, pons, medulla oblongata, and spinal cord. Hematoxylin and eosin stain revealed no viral inclusion bodies. Immunohistochemistry using a monoclonal antibody against nucleoprotein of canine distemper virus (CDV-NP) revealed a number of CDV-NP-positive granular deposits in the cytoplasm and cell processes of the degenerating or intact neurons. The present paper is a first report of spontaneously occurred morbillivirus infection in a dolphin at the Pacific Ocean around Japan. (+info)
Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.
Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature. (+info)
Comparative analysis of evolutionary mechanisms of the hemagglutinin and three internal protein genes of influenza B virus: multiple cocirculating lineages and frequent reassortment of the NP, M, and NS genes.
Phylogenetic profiles of the genes coding for the hemagglutinin (HA) protein, nucleoprotein (NP), matrix (M) protein, and nonstructural (NS) proteins of influenza B viruses isolated from 1940 to 1998 were analyzed in a parallel manner in order to understand the evolutionary mechanisms of these viruses. Unlike human influenza A (H3N2) viruses, the evolutionary pathways of all four genes of recent influenza B viruses revealed similar patterns of genetic divergence into two major lineages. Although evolutionary rates of the HA, NP, M, and NS genes of influenza B viruses were estimated to be generally lower than those of human influenza A viruses, genes of influenza B viruses demonstrated complex phylogenetic patterns, indicating alternative mechanisms for generation of virus variability. Topologies of the evolutionary trees of each gene were determined to be quite distinct from one another, showing that these genes were evolving in an independent manner. Furthermore, variable topologies were apparently the result of frequent genetic exchange among cocirculating epidemic viruses. Evolutionary analysis done in the present study provided further evidence for cocirculation of multiple lineages as well as sequestering and reemergence of phylogenetic lineages of the internal genes. In addition, comparison of deduced amino acid sequences revealed a novel amino acid deletion in the HA1 domain of the HA protein of recent isolates from 1998 belonging to the B/Yamagata/16/88-like lineage. It thus became apparent that, despite lower evolutionary rates, influenza B viruses were able to generate genetic diversity among circulating viruses through a combination of evolutionary mechanisms involving cocirculating lineages and genetic reassortment by which new variants with distinct gene constellations emerged. (+info)