DNA sequence evidence for polymorphic forms of human serum amyloid A (SAA)

Serum amyloid A (SAA) is an acute-phase reactant and precursor to amyloid A protein, the major constituent of the fibril deposits of reactive amyloidosis. The factors determining whether the 104-amino acid SAA molecule is converted into the 76-amino acid amyloid A protein and deposited as fibrils are not known. As an initial step toward investigating the possibility that a particular primary structure of SAA is involved in amyloid formation, we have cloned and determined the nucleotide sequence of human SAA-specific cDNAs. The first clone, selected using an oligonucleotide probe, was shown to encode the signal peptide and amino-terminal region of SAA. The cDNA of this clone served as probe in the selection of two distinct, full-length SAA cDNAs, initially differentiated by the presence (pSAA21) or absence (pSAA82) of a PstI site in the coding sequence. The complete nucleotide sequence of pSAA82 cDNA was determined. Since there appear to be multiple human SAA alleles, it is conceivable that their differential expression is important to amyloid formation.     

γ-Aminobutyric Acid Receptor Complex of Insect CNS: Characterization of a Benzodiazepine Binding Site

The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity-labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties.     

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Summary We report the complementation of a genetic lesion in the genome of Erwinia carotovora subsp. carotovora (Ecc), a pathogenic bacterium that incites soft rot of plants. A Sau3AI genomic library of Ecc was constructed using the conjugal cosmid pLAFR-3 as a vector. Sixteen cosmid clones encoding various plant tissue-degrading enzymes were identified, including a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. We detected a mutant of Ecc with no proteolytic activity following transposon mutagenesis with an unstable Tn5-carrying plasmid. Conjugal transfer of the protease-encoding cosmid to this mutant restored near-wildtype extracellular protease production. Further manipulation and study of genes encoding pathogenic determinants in Ecc will be possible using this system.     

The influence of subterranean termites on the hydrological characteristics of a Chihuahuan desert ecosystem

Summary Rainfall simulation at an average intensity of 124 mm·h^-1 was used to compare infiltration and run off on arid areas where subterranean termites had been eliminated four years prior to the initiation of the study (termite free) with adjacent areas populated by subterranean termites (termites present). Infiltration rates on termite free plots with less than 5% perennial plant cover were significantly lower 51.3±6.8 mm·h^-1 than rates on comparable termites present plots 88.4±5.6 mm·h^-1. On plots centered on Larrea tridentata shrubs, there were no differences in infiltration rates with or without termites. Plots with shrub cover had the highest infiltration rates 101±6 mm·h^-1. Highest run-off volumes were recorded from termite free <5% grass cover plots and the lowest from plots with shrubs. There were no differences in suspended sediment concentrations from termites present and termite free plots. Average bed load concentration was more than three times greater from termite free, <5% cover plots than from termites present, <5% cover plots.The reduction in infiltration, high run-off volumes and high bedloads from termite free areas without shrub cover is related to increased soil bulk density resulting from the collapse of subterranean galleries of the termites that provide avenues of bulk flow into the soil. Subterranean termites affect the hydrology of Chihuahuan desert systems by enhancing water infiltration and retention of top soil. The presence of a shrub canopy and litter layer cancels any effect of subterranean termites on hydrological parameters. Since approximately 2/3 of the area is not under shrub canopies, subterranean termites are considered to be essential for the maintenance of the soil water characteristics that support the present vegetation.     

Soil respiration of Alaskan tundra at elevated atmospheric carbon dioxide concentrations

Summary CO₂ efflux from tussock tundra in Alaska that had been exposed to elevated CO₂ for 2.5 growing seasons was measured to assess the effect of long- and short-term CO₂ enrichment on soil respiration. Long-term treatments were: 348, 514, and 683 μll⁻¹ CO₂ and 680 μll⁻¹ CO₂+4°C above ambient. Measurements were made at 5 CO₂ concentrations between 87 and 680 μll⁻¹ CO₂. Neither long- or short-term CO₂ enrichment significantly affected soil CO₂ efflux. Tundra developed at elevated temperature and 680 μll⁻¹ CO₂ had slightly higher, but not statistically different, mean respiration rates compared to untreated tundra and to tundra under CO₂ control alone.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Polyester Wadding for Specimen Orientation During Embedding in Methacrylates

Polyester fibers are not dissolved by either glycol methacrylate or methyl methacrylate. Commercial polyester wadding is consequently an advantageous material to use in getting precise orientation of tissue specimens during embedding in methacrylate.     

Evolution of karyotypic abnormalities and C-MYC oncogene amplification in human colonic carcinoma cell lines

Cell lines (COLO 320 DM and COLO 320 HSR), established from a human neuroendocrine tumor, contain an amplified cellular oncogene (c-myc). We have previously shown that the homogeneously staining regions (HSRs) of a marker chromosome in the COLO 320 HSR cells that evolved in culture from COLO 320 DM cells contain amplified c-myc. Molecular hybridization in situ has now been used to demonstrate that the HSRs are on both arms of what was once an X chromosome. We also show that amplified c-myc copies are present in the isolated double minute chromosomes (DMs) of the COLO 320 DM cells that were characteristic of the tumor cells initially established from the patient. The results suggest that the amplified c-myc appeared first as DMs and was subsequently transposed to engender HSRs on an X chromosome. The initial COLO 320 tumor cell may have acquired two early replicating (i.e., active) X chromosomes and lost the late replicating (i.e., inactive) X.     

Radiosensitivity of peripheral blood lymphocytes in autoimmune disease

The proliferation of peripheral blood lymphocytes, cultured with Con A, can be inhibited by ionizing radiation. Lymphocytes from patients with conditions associated with autoimmunity, such as rheumatoid arthritis, systemic lupus erythematosus and polymyositis, are more radiosensitive than those from healthy volunteers or patients with conditions not associated with autoimmunity. The nuclear material isolated from the lymphocytes of patients with autoimmune diseases is, on average, lighter in density than the nuclear material from most healthy controls. This difference in density is not related to increased sensitivity to ionizing radiation but the degree of post-irradiation change in density (lightening) is proportional to the initial density, i.e. more dense nuclear material always shows a greater upward shift after radiation. The recovery of preirradiation density of nuclear material, 1 h after radiation exposure, taken as an indication of DNA repair, correlates with the radiosensitivity of lymphocyte proliferation (Con A response); failure to return to pre-irradiation density being associated with increased sensitivity of proliferative response. These results require extension but, taken with previously reported studies of the effects of DNA methylating agents, support the idea that DNA damage and its defective repair could be important in the aetio-pathogenesis of autoimmune disease.