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Biomedical subjects

L K Riley

Publications and source records attributed to L K Riley.

77 records · Page 5Linked to original sources
PubMed1986

A method for biotinylating oligonucleotide probes for use in molecular hybridizations.

A new method is described for biotinylation of oligonucleotide probes for use in molecular hybridization reactions. Biotin-11-dUTP residues were added enzymatically, using terminal deoxynucleotidyl transferase, to the 3' terminus of a synthetic oligonucleotide prepared from the known nucleotide sequence for adenosine deaminase. The biotinylated probe was hybridized to DNA and mRNA selectively immobilized on nitrocellulose and detected by sequential incubation of the nitrocellulose membrane with avidin and biotinylated polyalkaline phosphatase, followed by colorimetric development. The biotinylated oligonucleotide probe proved useful for the qualitative detection of complementary DNA and mRNA sequences but was unsatisfactory for quantitative determinations using reflective densitometry.

Biotin↗
PubMed1984

Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes.

Bovine polymorphonuclear leukocytes (PMNs) were found to be significantly more bactericidal than human PMNs against a smooth-intermediate strain of Brucella abortus (45/0), whereas there was no difference in bactericidal activity of the two kinds of PMNs against a rough strain of B. abortus (45/20). Electron microscopy of thin sections of PMNs revealed that both strains of B. abortus were readily ingested; however, the extent of degranulation was significantly less than in PMNs incubated with an extracellular parasite, Staphylococcus epidermidis. Amounts of myeloperoxidase and lactoferrin released through exocytosis by PMNs incubated with S. epidermidis were 4.7- and 1.2-fold greater, respectively, than those released from PMNs incubated with B. abortus 45/0. When azurophil and specific granules were isolated after incubation of PMNs with either B. abortus 45/0 or S. epidermidis, results showed that the extent of degranulation by both types of granules was greater in PMNs incubated with S. epidermidis than in those incubated with B. abortus 45/0. Amounts of degranulation by azurophil and specific granules were similar in PMNs incubated with either the smooth-intermediate strain 45/0 or the rough strain 45/20. Degranulation was not stimulated when glutaraldehyde-killed strain 45/0 was substituted for viable cells. These data suggest that B. abortus does not stimulate an effective level of degranulation after ingestion, as observed with extracellular parasites, and that the smooth intermediate strain 45/0 is more resistant to intraleukocytic killing system than the rough strain 45/20.

Animals↗
PubMed1984

Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus.

The microbicidal activities of freeze-thaw and high-salt extracts of human and bovine polymorphonuclear leukocyte (PMN) granules were tested against a smooth intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus which differ in virulence and survival within PMNs. Freeze-thaw extracts of human PMN granules were more brucellacidal than high-salt extracts when supplemented with hydrogen peroxide (H2O2) and potassium iodide (KI), whereas the opposite was found with freeze-thaw and high-salt extracts of bovine PMN granules. There was no oxygen-independent killing of either the smooth or rough strain of B. abortus by amounts of granule extracts which caused 100% killing of a deep rough mutant (Re) of Salmonella typhimurium. The oxygen-dependent brucellacidal activity of granule extracts was dependent on concentrations of myeloperoxidase (MPO) units, H2O2, and KI. Maximal brucellacidal activity was observed at pH 5.5 to 6.0. The smooth strain, 45/0, was more resistant to oxygen-dependent killing by granule extracts than was the rough strain, 45/20. Granule extracts were more brucellacidal than purified MPO at equivalent levels of MPO enzyme units, suggesting that at least one other reaction enhances killing by the MPO-H2O2-I- system.

Animals↗
PubMed1983

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development.

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.

Adolescent↗