Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Immunoglobulin G antibody bound to the C1q subcomponent of human complement exhibits segmental flexibility

The rotational dynamics of rabbit immunoglobulin G (IgG) anti-dansyl antibodies bound to the C1q subcomponent of human complement were studied by nanosecond fluorescence spectroscopy. Deconvoluted anisotropy decays of IgG-C1q mixtures were fitted to a two-exponential expression and were corrected for the effects of unbound IgG, which was determined with an analytical ultracentrifuge. Compared with the anisotropy parameters for free IgG, the pre-exponential weighting factors and the short correlation time of the C1q-bound antibody were nearly unchanged, and the long correlation time increased by only about 45 nanoseconds. These results, together with rotational diffusion calculations, indicate that the Fab arms of the C1q-bound antibody exhibited considerable flexibility. This finding may have biological relevance because it suggests that C1q can bind to the Fc segments of IgG molecules anchored in an immune complex, even though the angles between the two Fab arms of the different antibodies may vary. The results of this study also support our earlier interpretation that both the short and long correlation times of IgG principally represent flexible motions of the Fab segments.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.     

Leishmania braziliensis: effects of bacteria (Staphylococcus aureus and Pasteurella multocida) on the developing cutaneous leishmaniasis lesion in the golden hamster

Experimentally induced lesions of cutaneous leishmaniasis and the effect of concurrent bacterial infection on the development of these lesions were studied in the golden hamster. Male outbred golden hamsters received intradermal injections at the base of the tail with approximately 10(7) promastigotes of Leishmania braziliensis panamensis, or promastigotes combined with Staphylococcus aureus or Pasteurella multocida or both, bacteria only, or sterile Eagle's minimal essential medium (MEME). The size of the resulting lesions was measured at least twice each week. Hamsters were killed at postinoculation Days 6, 13, 20, 27, 41, or 48, and each lesion was measured, aseptically excised, and bisected; half was used for bacteriologic culture and the other half was prepared for light microscopic examination. Lesions resulting from L. b. panamensis alone progressed from initial erythema to a granulomatous nodule and finally to a necrotic granuloma, often capped by a crateriform ulcer. Lesions resulting from a suspension of L. b. panamensis with added S. aureus or S. aureus and P. multocida, were initially larger, more erythemic and contained a greater proportion of neutrophils up to postinoculation Days 14-21 than did lesions resulting from L. b. panamensis alone. Concurrent infections with bacteria such as S. aureus and P. multocida had little effect on the development of ulcerating characteristics of lesions, but when S. aureus was present it appeared to enhance the severity of the early lesions. Between postinoculation Days 14-28, lesions produced by L. b. panamensis, with or without added bacteria had similar developmental progression of sufficient size for optimal testing of antileishmanial compounds.