Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism. VI. Enzymatic studies of two mutants unable to convert inosinic acid to adenylic acid

Ade^–H and ade^–I are two auxotrophic mutants of Chinese hamster ovary (CHO-K1) cells which specifically require adenine as the purine source to grow. The enzymatic defects of these mutants were examined in cell-free extracts. It was found that ade^–H did not have any detectable adenylosuccinate synthetase activity and ade^–I was defective in the adenylosuccinate lyase enzyme. The relevance of adenine-requiring mutants to the study of the regulation of purine metabolism in mammalian cells is discussed.This work was supported by research grants from the National Institute of Aging (AG00029) and the National Foundation, March of Dimes (1-423), and by a contract from the Center for Toxicological Research, Food and Drug Administration (72-213). David Patterson is a recipient of a Research Career Development Award from the National Institute of Arthritis, Metabolic and Digestive Diseases (AM00044).Contribution (No. 218) from the Eleanor Roosevelt Institute for Cancer Research.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosphingolipids of normal and leukemic human leukocytes

Summary We have reviewed the studies on neutral glycosphingolipids and gangliosides of normal and leukemia human leukocytes. In this review, we examine (a) the glycosphingolipid composition of various leukocyte populations, (b) the differences in glycosphingolipids found among subsets of these cells, (c) the possible use of these compounds as markers of differentiation, and (d) the changes in glycosphingolipid composition that occur with leukemogenesis.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.     

The interaction of chlordiazepoxide and sodium valproate in the nucleus accumbens of the rat

Benzodiazepines are known to facilitate GABA-ergic transmission at synaptic sites, while sodium valproate is an anticonvulsant drug which is reported to elevate GABA levels in the brain. In order to determine whether these two drugs interact functionally at GABA receptor sites, graded doses of chlordiazepoxide (CDZ) and sodium valproate were injected bilaterally into the nucleus accumbens and their effect on the dopamine (DA)-induced stimulation of motor activity was studied. Both of these compounds, as well as GABA, produced an inhibition of the hyperactivity induced by the bilateral injection of DA into the nucleus accumbens. Bicuculline, the GABA receptor antagonist, blocked the effect of CDZ on the DA-induced hyperactivity. A low dose of CDZ (2 μg), which by itself did not significantly inhibit the effect of DA, potentiated the inhibition of the hyperactivity produced by valproate. These results suggest that CDZ and sodium valproate can interact functionally at GABA-ergic sites in the central nervous system.     

Field studies of the relationship between herbivore damage and tannin concentration in bracken (Pteridium aquilinum Kuhn)

Summary The acceptance of secondary plant metabolites as herbivore deterrents rests primarily on their deleterious effects on herbivores. Efforts to demonstrate differential fitness in natural plant populations with varying concentrations of tannin have failed, since coevolved plant predators may physiologically or behaviorally circumvent the defense, which results in apparently equal amounts of damage to defended and undefended individuals. In this study, two approaches were used to overcome this difficulty. 1) Theoretically, more energy should be allocated to the defense of parts which contribute more heavily to the plant's fitness. Bracken fern clones produce fronds throughout the growing season. Fronds which are produced early should be more heavily defended than late-emerging fronds which will return less photosynthate per unit cost of production. The results of this study do not support this prediction; it appears that the production of tannin is more closely linked to environmental factors such as water stress than to date of frond emergence. Fronds which emerged in August contained as much tannin as fronds which emerged in May. 2) By recording the temporal occurrence of herbivore damage in bracken ferns, it was found that in fronds which escaped attack until after reaching maturity there was a significant negative correlation between tannin concentration in the frond and the amount of damage experienced. This result supports the generally accepted assumption that herbivory has been a selective force in the evolution of tannin as a defensive substance.