H-2-linked Ir gene control of T cell recognition of the Sm nuclear autoantigen and the aberrant response of autoimmune MRL/Mp-+/+ mice

We have examined T cell recognition of the nuclear autoantigen Sm. Rabbit Sm-primed cells from autoimmune MRL/Mp-+/+ (+/+) mice and from all normal strains tested were able to proliferate to rabbit Sm in vitro. In contrast, the reactivity of normal strains to Sm of murine origin was genetically restricted; only H-2f strains B10.M and A.CA, and H-2s strains B10.S and A.SW could recognize mouse Sm, suggesting that responsiveness to mouse Sm was under the control of H-2-linked Ir genes. Although five Iak-bearing normal strains (B10.A, B10.A(2R), B10.BR, A/Sn, and CBA) did not recognize mouse Sm, autoimmune +/+ (Iak) mice were responders. The responsiveness of the +/+ mice to Sm was probably not due to differences in their Iak region, compared with other strains, because the Iak region of normal strains and the autoimmune +/+ strain were indistinguishable by interstrain MLC, immune response gene product function, and recognition by anti-Iak mAb. Inhibition of Sm-induced proliferation by mAb demonstrated that T cells from autoimmune +/+ mice, responder normal strains, and nonresponder normal strains recognized rabbit and mouse Sm in the context of I region-encoded products. The T cell response to Sm antigen in normal mice is therefore Ia region restricted and, for the murine antigen, under Ir gene control. Autoimmune mice that spontaneously make anti-Sm antibodies (+/+) also perceive Sm in an Ia-restricted manner, but their responder status abrogates H-2-linked Ir gene control.     

Evidence of DNA repair in organ cultures of hamster tracheal epithelium following exposure to gas phase singlet oxygen

Autoradiographic identification of unscheduled DNA synthesis (UDS) in short-term organ culture of hamster tracheal epithelium has been used as a predictive test for mutagenic and/or carcinogenic compounds. Tracheal explants were treated for 2 h with singlet delta oxygen plus [3H]thymidine. Silver grains over the nuclei of epithelial cells from the superficial layer of the mucosa were observed, indicating UDS. Control cultures, exposed to the gas phase without singlet oxygen, failed to elicit UDS.     

Mitochondrial Changes in Harvested Carnation Flowers (Dianthus caryophyllus L.) during Senescence

Harvested carnation (Dianthus caryophyllus L.) flowers wereplaced in either a preservative solution or deionized waterand monitored through senescence during which time flower freshweight was measured as well as production of ethylene and CO₂.Flower fresh weight, ethylene, and CO₂ levels increased as theflowers aged, but fresh weight and CO₂ levels fell once flowersbegan to senesce regardless of holding solution. Preservative-treatedflowers senesced at a slower rate than deionized water-treatedflowers. The amount of ADP phosphorylated to ATP per oxygenatom consumed, using mitochondria isolated from petal tissueprovided with either succinate or malate as substrates, wasfound to increase as flowers senesced and then to decrease inthe later stages of senescence. Respiratory control ratios withsuccinate as the substrate did not change appreciably untilthe final stages of senescence white respiratory control valuesusing malate showed greater variation but no consistent patternrelative to the progress of senescence. Cyanide-resistant respirationwas noted with isolated mitochondria oxidizing either substrate,but no correlation between cyanide-resistant respiration andsenescence could be found. (Received July 10, 1984; Accepted April 16, 1985)     

Bacteriophage phi X174 A protein cleaves single-stranded DNA and binds to it covalently through a tyrosyl-dAMP phosphodiester bond.

The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end. To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with [alpha-32P]dATP to form the complex of A protein covalently linked to single-stranded DNA. The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction). The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide. The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine. These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.     

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.     

Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure

To aid in the interpretation of the 3.5 A resolution electron density map of glutamine synthetase (GS) from Salmonella typhimurium, the nucleotide sequence of the gene coding for this enzyme has been determined. The predicted sequence of 468 amino acids (Mr = 51,628) has been compared to the sequence and sequence fragments reported by others for GS of Anabaena and Escherichia coli. The homology between the pairs of sequences is sufficiently strong to suggest that the overall three-dimensional structures of the three GS are similar. The predicted positions of alpha helices are in moderately good agreement with the electron-density map.     

Molecular basis for the special properties of proteins and enzymes from Halobacterium marismortui

Abstract Three proteins from Halobacterium marismortui , malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.     

The design, synthesis, and crystallization of an alpha-helical peptide

Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.     

Bifunctionality and pseudoisozymes of pyruvate kinase from codfish muscle

Pyruvate kinase has been purified from codfish muscle. The ratio of phosphotransferase and oxalacetate decarboxylase activities remains relatively constant throughout purification steps. These two activities are dependent as well as sensitive to sulfhydryl reagents. In the presence of dithioerythritol, only one molecular form of pyruvate kinase is detected. However, the enzyme exists as four pseudoisozymes in the presence of 2-mercaptoethanol. The pseudoisozymes of codfish pyruvate kinase are interconvertible under the influence of sulfhydryl reagents.     

Glycopeptides of the Type-Common Glycoprotein gD of Herpes Simplex Virus Types 1 and 2

We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [³H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [³H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [³H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [³⁵S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2.