ABSTRACT
32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible.
Subject(s)
Chromatography, Ion Exchange/methods , Chromatography, Thin Layer/methods , DNA Adducts/analysis , Animals , Female , Mice , Phosphorus RadioisotopesABSTRACT
Five bactericidal peptides (chicken heterophil peptides CHP1 and CHP2; turkey heterophil peptides THP1, THP2, and THP3) were purified from avian heterophil granules. All peptides were cationic and rich in cysteine, arginine, and lysine. The complete amino acid sequence, consisting of 39 amino acids, was determined for CHP1. This peptide had a molecular weight of 4481 as determined by mass spectrometry. Partial NH2-terminal amino acid sequences were obtained for the remaining peptides. Both chicken peptides and THP1 shared sequence homology at 22 residues and a cysteine motif which was similar to that of bovine beta-defensins. THP2 and THP3 were homologous to each other but were not homologous to the other three and had a unique cysteine motif. Peptides CHP1, CHP2, and THP1 killed Staphylococcus aureus and Escherichia coli in vitro, whereas THP2 and THP3 killed only S. aureus in vitro.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Antimicrobial Cationic Peptides , Avian Proteins , Defensins , Leukocytes/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Chickens , Escherichia coli/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data , Staphylococcus aureus/drug effects , TurkeysABSTRACT
Chicken leg muscle parvalbumin was digested with cyanogen bromide or trypsin or trypsin after citraconylation. Peptides isolated by reverse phase HPLC at pH 7.0 were subjected to acid hydrolysis and amino acid analysis and, in some cases, sequencing. The chicken muscle parvalbumin amino acid sequence has ca. 80% sequence identity with alpha-type parvalbumins from mammalian (rabbit, human and rat) muscle. By contrast, the chicken thymus parvalbumin ("avian thymic hormone") sequence is very similar to reptile (turtle, salamander and frog) muscle beta-type parvalbumins. We hypothesize that the evolutionary appearance of the warm-blooded reptiles was accompanied by recruitment of the beta parvalbumin isozyme for promotion of lymphocyte maturation.
Subject(s)
Biological Evolution , Muscles/physiology , Parvalbumins/genetics , Thymus Gland/physiology , Amino Acid Sequence , Animals , Chickens , Humans , Mammals , Molecular Sequence Data , Parvalbumins/chemistry , Reptiles , Sequence Homology, Nucleic AcidABSTRACT
Avian thymic hormone (ATH), a parvalbumin with immunomodulating properties, was used as antigen for development of a hybridoma cell line. Hybridoma screening by enzyme-linked immunosorbent assay (ELISA) led to the culture, purification, and characterization of an hybridoma, designated 4A6, which secretes IgG1 that is specific for ATH. The monoclonal antibody (MAb) was characterized by techniques involving indirect competitive and non-competitive ELISA, Western Blot analysis, and immunohistological staining. The MAb was highly specific for ATH with no cross-reactivity to other chicken tissue extracts or commercially available parvalbumins.
Subject(s)
Antibodies, Monoclonal/immunology , Chickens/immunology , Parvalbumins/immunology , Thymus Gland/immunology , Animals , Enzyme-Linked Immunosorbent Assay , Hybridomas/immunology , Immunoenzyme Techniques , Immunoglobulin G/immunology , Organ Specificity , Parvalbumins/analysis , Species Specificity , Thymus Gland/chemistryABSTRACT
Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.
