Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness.

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C(+)Ly-6G(-) and they downmodulated T-cell activation, recruited T(reg) cells, and dramatically downregulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C(-)Ly-6G(+) and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C(+)Ly-6G(+) cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.

Urokinase binding and receptor identification in cultured endothelial cells.

Recombinant human single-chain urokinase (rscu-PA), two-chain urokinase (tcu-PA), and diisopropyl-fluorophosphate-treated tcu-PA (DFP-tcu-PA) bound to cultured human and porcine endothelial cells in a rapid, saturable, dose-dependent and reversible manner. Analysis of specific binding results in cultured human umbilical vein endothelial cells (HUVECs) gave the following estimated values for Kd and Bmax: 0.57 +/- 0.08 nM (mean +/- S.E.) and 188,000 +/- 18,000 sites/cell for 125I-labeled rscu-PA; 0.54 +/- 0.10 nM and 132,000 +/- 23,900 sites/cells for 125I-labeled tcu-PA; 0.89 +/- 0.14 nM and 143,000 +/- 30,300 sites/cell for 125I-labeled DFP-tcu-PA, respectively. Values for Kd were similar for primary and subcultured (six passages) HUVECs, but Bmax values were lower in subcultured HUVECs. Similar Kd values were found in cultured porcine endothelial cells; however, Bmax values varied depending on the endothelial cell type. All 125I-labeled urokinase forms yielded similar cross-linked approximately 110-kDa ligand-receptor complexes with cultured HUVECs, and 125I-labeled DFP-tcu-PA bound to a single major approximately 55-kDa protein in whole-cell lysates (ligand blotting/autoradiography), suggesting the presence of a single major approximately 55-kDa urokinase receptor in cultured HUVECs. The approximately 55-kDa urokinase receptor, isolated from several separate batches of cultured HUVECs (3-5 micrograms of protein, approximately 1 x 10(9) cells), by ligand affinity chromatography, exhibited the following properties: retained biologic activity as evidenced by its ability to bind 125I-labeled rscu-PA by ligand blotting/autoradiography and formation of a cross-linked 125I-labeled approximately 110-kDa rscu-PA-receptor complex; single-chain approximately 55-kDa protein, following reduction; complete conversion to and formation of a single major deglycosylated approximately 35-kDa protein, following treatment with N-glycanase.

Transformation of DNA sequence data into geometric symbols.

Comparative analysis of related DNA sequences has been simplified by the transformation of data in the standard A, G, C, T format into a set of geometric symbols that promote pattern recognition. Previously, comparing more than 2 or 3 sequences simultaneously has been difficult because of the monotonous patterns established by letters. Here 33 sequences are simultaneously compared to demonstrate the ease with which nucleotide substitutions are accurately identified. This has been accomplished by writing a Word-Perfect macro program to facilitate this transformation. Since this word processing program is widely used, performing this kind of analysis is readily achievable in most laboratories involved in DNA sequence analysis.

Filler DNA is associated with spontaneous deletions in maize.

We have determined the structure of five spontaneous deletions within the maize waxy (Wx) gene. Of these, four were found in spontaneous wx mutants (wx-B, wx-B1, wx-B6, wx-C4) and include exon sequences; the fifth is restricted to an intron and represents a restriction fragment length polymorphism of a nonmutant allele (Wx-W23). The deletions, which range in size from 60 to 980 base pairs (bp), cluster in a G+C-rich region of approximately 1000 bp that is capable of forming stable secondary structures. Most striking is our finding that all of the alleles have DNA insertions (filler DNA) of 1-131 bp between the deletion endpoints. For three of the five deletions, the filler DNA and sequences at the deletion termini appear to be derived from sequences near one deletion endpoint. A previously reported spontaneous deletion of the maize bronze gene (bz-R) also contains filler DNA. The association of filler DNA with maize deletion endpoints contrasts dramatically with the rarity of similar events in animal germ-line and bacterial mutations.

An RFLP adjacent to the maize waxy gene has the structure of a transposable element.

Two maize inbred lines harbor non-mutant waxy (Wx) genes that display restriction fragment length polymorphism (RFLP) upstream from the start of Wx transcription. Sequencing of this region in the two strains revealed a DNA insertion with the structural features of a transposable element. The insertion is 316 bp in length, has 15 bp imperfect inverted repeats and is flanked by a 5 bp direct repeat generated upon insertion. Sequences homologous to this insertion are present in multiple copies in maize and its relatives teosinte and Tripsacum but not in the more distantly related dicot tobacco. Finally, this element is not homologous with any previously described maize DNA insertion.

Insulin resistance in total lipodystrophy: evidence for a pre-receptor defect in insulin action.

The cause of insulin resistance in lipodystrophic diabetes is unknown but has generally been ascribed to dysfunction at either the receptor or post receptor level. In a 14 year-old girl with total acquired lipodystrophy, subcutaneous and intravenous insulin requirements approximated 600 units daily. However, circulating total and free insulin levels were not increased, and during testing by the euglycemic clamp method, the glucose response to increasing free insulin concentrations was within the range found in eight subjects with insulin-dependent diabetes. Insulin clearance during the euglycemic clamp was 43, 98, 115, and 116 mL/kg/min at each of four insulin infusion rates compared to means of 13, 13, 12, and 11 in the control subjects with diabetes. No detectable degrading activity was present in serum, and serum inhibited insulin degradation normally. Binding of insulin to IgG, IgM, and IgE was not increased, insulin binding to monocytes and erythrocytes was not sufficiently abnormal to account for the the insulin resistance, and insulin receptor increased insulin clearance or accelerated degradation of insulin by tissues.