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Biomedical subjects

R Nairn

Publications and source records attributed to R Nairn.

At least 37 records · Page 2Linked to original sources
PubMed1987

Pseudomembranous colitis: a lethal complication of Hirschsprung's disease unrelated to antibiotic usage.

Seventy-two new cases of Hirschsprung's disease were seen between 1980 and 1985. Twenty-six patients (36%) developed the clinical features of enterocolitis and, of nine patients who died, colitis was the immediate cause of death in six. Histologic material was available from 20 patients with colitis; this showed nonspecific inflammation typical of Hirschsprung's colitis in 13 cases but seven had pseudomembranous colitis (PMC). Five of the patients with PMC had not recently been exposed to antibiotics. PMC was responsible for three of the six deaths from colitis. All patients with Hirschsprung's disease who develop signs of colitis should have stool testing for Clostridium difficile toxin and should be treated with an antibiotic active against Clostridium difficile.

Bacterial Proteins↗
PubMed1985

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor.

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.

Affinity Labels↗
PubMed1985

DNA sequence of the mouse H-2Dd transplantation antigen gene.

The inbred BALB/c mouse has three transplantation antigens, H2-Kd, H2-Ld, and H2-Dd. We present the complete nucleotide sequence of the H2-Dd gene as well as 777 residues of previously unpublished H-2Dd protein sequence. These data complete the sequences of all the BALB/c transplantation antigen genes and permit detailed comparison with each other and with their counterparts from the inbred C57BL/10 mouse. Transplantation antigens may differ from one another by as much as 5%-15% of their amino acid sequence for the external domains. These extensive differences may arise by gene conversion. The H-2D region of the BALB/c mouse encodes the H2-Dd and the H2-Ld genes. Serologic data suggest that at least two additional transplantation antigen molecules, H2-Rd and H2-Md, are encoded in the H-2D region of the major compatibility complex. Paradoxically, gene cloning studies have only identified the H2-Dd and the H2-Ld genes in the H-2D region. A complete DNA sequence of the H2-Dd gene shows that a variety of alternative splice sites exist throughout the gene, which may lead to additional gene products and may explain the multiplicity of H-2D-encoded polypeptides.

Amino Acid Sequence↗
PubMed1985

Importance of the COOH terminal of angiotensin in antigenicity and in the formation of an antigen-containing complex with cellular membrane structures.

To more carefully determine how a peptide antigen interacts with the antigen-presenting cell (APC), we have begun an analysis of the fate of APC-associated peptide antigens. These studies have shown that a stable cell-bound form of APC-associated peptide exists, which is a complex of the peptide with surface membrane structures (peak A). In the experiments described here, we have begun to examine the chemical mechanism of this peak A complex formation. By modifying either the carboxyl terminal or amino terminal group of the octapeptide antigen angiotensin II we have established that the terminal carboxyl group, but not the terminal amino group, was critical for forming the peak A complex with APC membrane structures. In addition, blocking the carboxyl but not the amino terminal dramatically reduced the antigenicity of the peptide for AII-immune T cell in vitro proliferation. These results show that the carboxyl terminal of AII is essential for both peak A formation and antigenicity, and suggest that peak A is critical for antigen presentation to T cells.

Acetylation↗
PubMed1984

Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.

Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography. The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with [3H]fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material. Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity. Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated. The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered. The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences. After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3). Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine. The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe. Butanol extracts from E. coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E. coli was found. Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates. These results demonstrate that the NH2-terminal formyl peptides produced by E. coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.

Animals↗
PubMed1984

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.

Amino Acid Sequence↗
PubMed1984

Macrophage processing of peptide antigens: identification of an antigenic complex.

In this report, we present data from our examination of the fate of the octapeptide antigen angiotensin II (AII) after processing by guinea pig macrophages. Macrophages were cultured with [125I]-AII or [3H]-AII for various times, and the AII products in the culture supernatant fluid and solubilized cell lysate (macrophage-associated AII) were analyzed by chromatography on Sephadex G-25. With [3H]-AII, both the cell-associated and cellfree AII was catabolized to three distinct peptide fragments: Peak B, containing seven to eight amino acids; Peak B1, with five to six amino acids; and Peak C, containing four to five amino acids. Surprisingly, in the cells another major form of AII was obtained, Peak A, which showed an m.w. of 50,000 to 70,000. Formation of Peak A was blocked by inclusion of non-radiolabeled AII into the incubation mixture, and could be formed by repulsing fresh macrophages with isolated Peaks B and B1, and to only a minor extent with Peak C. Peak A was formed within an hour of culture of macrophages with [3H]-AII, and remained stably cell-associated through a day of culture. In a pulse-chase type of experiment, it was shown that macrophages rapidly exocytose cell-associated Peaks B and C, and Peak B1 less rapidly, whereas Peak A is retained and is the predominant cell-associated form of AII after a day in culture. Incubation of [3H]-AII with isolated macrophage plasma membranes also resulted in Peak A formation, showing that the complex can be formed by a membrane event not requiring cellular uptake of peptide. The complex of AII in Peak A was acid-stable, and approximately 20% of Peak A was recovered after boiling in SDS. Because Peak A is the primary form of AII retained by macrophages, it was important to determine if this complex was antigenic for T cells. AII-immune T cells cultured with Peak A showed significant proliferation that was specific for AII. Thus, Peak A appears to represent a stable complex of AII with macrophage membrane structures that provide an immunologically relevant form of the antigen. These results are discussed with respect to a pathway for macrophage processing of peptide antigens to form a stable membrane-bound complex that is important for T cell responses.

Amino Acid Sequence↗
PubMed1982

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens.

The H-2L-dm1 and H-2Ddm1 MHC antigens of the B10.D2 (H-2dm1) mutant mouse strain (formerly known as M504 or H-2da) have been compared to the H-2Ld and H-2Dd antigens of the B10.D2 (H-2d)mouse strain. Ldml and Ld are 45 000 Mr antigens and both are reactive with anti-H-2."28" (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the Ddm1 and Dd antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between Ldm1 and Ld is 80 percent. A newly defined antigen (mr = 39 000), designated gp39dm1, was found in glycoprotein extracts of the dm1 strain but not of the d strain. This antigen coprecipitates with Ldm1 but does not coprecipitate with Ddm1 indicating that it lacks the H-2.4 determinant. In comparison with Ldm1, gp39dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of Ldm1. Finally, peptide maps of the Ddm1 antigen show that the majority of its Arg peptides are identical to Dd Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to Dd peptides but to Ld and Ldm1 peptides. These data raise the possibility that the Ddm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.

Animals↗
PubMed1981

Amino acid sequence of cyanogen bromide fragment CN-C (residues 24-98) of the mouse histocompatibility antigen H-2Dd. A comparison of the amino-terminal 100 residues of H-2Dd, Dd, Kd, and Kb reveals discrete areas of diversity.

The amino acid sequence of the cyanogen bromide (CNBr) fragment extending from position 24 to position 98 of the H-2Dd murine histocompatibility antigen has been determined by using radiochemical microsequencing techniques. This 75-residue fragment (CN-C) is one of two glycopeptides generated by CNBr cleavage of the extracellular portion of the H-2Dd molecule (H-2Dd papain). Determination of this sequence completes the amino-terminal 100 residues of the H-2Dd molecule. The primary structure of CN-C was established by thrombin digestion of isolated CN-C and sequence determination of the three resulting peptides. The COOH-terminal met and its adjacent residue were determined by sequence analysis of a tryptic peptide which overlaps CNBr fragments C and b4 (residues 99-138). Alignment of the thrombic peptides was accomplished by NH2-terminal sequence analyses of CN-C and peptides generated by Staphylococcus aureus V8 protease digestion of CN-C. The sequence data presented here, together with that already given for H-2Dd [Nairn, R., Nathenson, S. G., & Coligan, J.E. (1980) Eur. J. Immunol. 10,495-503], allow a comparison of the NH2-terminal 100 residues of the Dd, Db, Kd, and Kb molecules. Discrete areas of diversity, in particular, one between residues 62 and 83, are obvious. Comparison over some 180 residues of the Dd and Kb molecules reveals a particularly close similarity between these products of a K and a D gene from widely disparate mouse strains.

Amino Acid Sequence↗
PubMed1980

Isolation, characterization and amino acid sequence studies of the cyanogen bromide fragments of the H-2Dd glycoprotein.

The papain-solubilized fragment of the H-2Dd antigen, representing the NH2-terminal 80% of the native glycoprotein, can be fragmented into five polypeptides using CNBr cleavage. These fragments have been isolated, characterized and subjected to amino acid sequence analysis using radiochemical microtechniques. It was possible to align these CNBr fragments by comparison of their NH2-terminal amino acid sequence with the known amino acid sequence of the H-2Kb molecule. Thus, the NH2-terminal fragment, D, contains 23 residues and is followed by a glycopeptide, fragment C, of 75 residues. Following fragment C are three fragments held together by disulfide bonds: fragment b4, which has 40 amino acid residues, a second glycopeptide, fragment b2, of 90 residues, and fragment b3, a peptide of approximately 55 residues which terminates at the site of papain cleavage. The complete amino acid sequence of the NH2-terminal CNBr fragment, D, has been determined, and this is presented here along with sequence data for the NH2-terminal 30 residues of each of the other four CNBr fragments of the papain fragment of H-2Dd. A total of 146 positions have been examined in the approximately 280-residue H-2Ddpapain molecule, and a total of 127 residues have been positively determined. Most of the unassigned positions may be tentatively assigned as Asp or Asn, the only amino acids so far not incorporated in radiolabeled form by our metabolic labeling procedures into the H-2Dd molecule. Comparison of the sequence data obtained for the H-2Dd molecule to data available for other histocompatibility antigens revealed homologies of approximately 90% with H-2Kb and H-2Ld, and approximately 75% with HLA-B7 and HLA-A2.

Amino Acid Sequence↗
PubMed1980

Primary structural studies of an H-2L molecule confirm that it is a unique gene product with homology to H-2K and H-2D antigens.

Radiochemical methodology has been used in the isolation and preliminary biochemical characterization of the murine H-2Ld major histocompatibility complex gene product. The radiolabeled molecule was isolated by immunoprecipitation from the glycoprotein fraction of detergent-solubilized H-2d tumor cells. Six major CNBr fragments were isolated from a papain fragment of this molecule; three of the fragments are connected by disulfide bonds. Due to the high degree of homology between major transplantation antigens, it was possible to align the fragments by comparison of their amino acid sequences to that of the H-2Kb gene product. Of the positions available for comparison between H-2Ld and H-2Kb, H-2Dd, and H-2Kd gene products, 61 out of 80 (78%), 45 out of 55 (82%), and 12 out of 15 (80%), respectively, are identical. Differences between the Ld and Kb and Dd molecules are distributed throughout the amino acid sequence. These data indicate that the H-2Ld gene product is a molecular species distinct from, but homologous to, the H-2K and H-2D gene products.

Amino Acid Sequence↗
PubMed1978

Structural studies of the H-2D products of the mouse mutant BALB/c-H-2Ddb and the parental strain BALB/cKh-H-2Dd.

The tryptic peptide profile characteristics of the H-2D glycoprotein, isolated by immunoprecipitation from the MHC mutant mouse strain BALB/c-H-2Ddb, were compared with those of the H-2D molecule from the parent strain BALB/cKh-H-2Dd. At each stage of purification these molecules exhibited identical biochemical properties and on peptide mapping we observed that the Ddb molecule showed no detectable peptide differences from the Dd molecule of the nonmutant parent. These data thus support the concept that the site of mutation in this mutant strain, although located in the D region of the MHC, is distinct from the gene coding for molecules bearing the H-2.4 private specificity.

Animals↗
PubMed1977

Characteristics of a human cell line transformed by DNA from human adenovirus type 5.

Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in otheruman cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 250 K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.

Adenoviruses, Human↗
PubMed1977

Effect of 2-deoxy-D-glucose on the cell-surface glycoproteins of hamster fibroblasts,.

1. Growth of baby hamster kidney (BHK) cells in medium containing 2-deoxy-D-glucose is retarded in direct proportion of the 2-deoxyglucose concentration. The severity of the effect is reduced in medium containing high relative concentrations of glucose. 2. 2-Deoxyglucose inhibits the incorporation of radioactivity from mannose, galactose, glucosamine, fucose and N-acetylmannosamine precursors into acid-insoluble cellular material. Incorporation of radioactively labelled leucine into protein is not affected by 2-deoxyglucose. 3. BHK cells grown in the presence of 2-deoxyglucose become less sensitive to the toxic action of certain plant lectins, ricin of Ricinus communis and Phaseolus vulgaris phytohaemagglutinin, which bind specifically to cell surface galactose and N-acetyl-galactosamine residues. By contrast, 2-deoxyglucose increased the sensitivity of BHK cells to the weak toxicity of concanavalin A, which binds to surface mannosides. Treated cells also become more agglutinable with concanavalin A. 4. Cell surface glycoprotein labelled by lactoperoxidase-catalysed iodination have been examined by dodecylsulphate-polyacrylamide gel electrophoresis. The radio-iodinated glycoprotein prepared from cells grown in medium containing 2-deoxyglucose migrate more rapidly than glycoproteins from cells grown in the absence of inhibitor.

Cell Division↗