A beta-linked mannan inhibits adherence of Pseudomonas aeruginosa to human lung epithelial cells

Adherence through carbohydrate-binding adhesins is an earlystep in colonization of the lung by gram-negative organisms,and because published data indicate that binding involves mannosegroups, we tested the ability of a ß-linked acetylmannan(acemannan) to inhibit adherence of Pseudomonus aeruginosa tocultures of human lung epithelial cells. Adherence of radiolabelledP.aeruginosa to A549 cells (a type II-like pneurnocyte line)increased linearly with the duration of the incubation. Acemannaninhibited adherence of bacteria, and the extent of inhibitionwas related to the concentration of the mannan. Inhibition requiredcontinued contact between acemannan and the target epithelialcells; cells washed free of acemannan no longer discouragedbacterial binding. Comparison of binding between seven strainsof P.aeruginosa indicated that fewer mucoid than non-mucoidbacteria adhered, but binding of either phenotype was inhibitedby acemannan. Mannose methyl -D-mannopyranoside, methyl ß-D-mannopyrannosideand dextran did not affect adherence of any of the non-mucoidstrains. Mannose inhibited adherence by one mucoid strain, butnot the other, indicating differences between strains of thesame phenotype. Since prior treatment of epithelial cells withconcanavalin A did not affect acemannan-induced inhibition ofbacterial adherence, we concluded that the inhibitory effectof acemannan probably does not involve mannose-containing receptors. bacterial-host interactions lung epithelium mucoid strains non-mucoid strains     

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.     

In vivo replication of carcinogen-modified rat liver DNA: increased susceptibility of O6-methylguanine compared to N-7-methylguanine in replicated DNA to S1-nuclease

In order to characterize rat liver DNA replicated $\text{in}\text{vivo}$ on a carcinogen-damaged template, the replicated DNA was treated with S₁-nuclease and the release of (¹⁴C)-dimethyl-nitrosamine induced 0⁶-methylguanine, a lesion associated with miscoding and N-7-methylguanine, a lesion that does not miscode were monitored. The results indicated that both the methylated guanines became susceptible to S₁-nuclease upon replication. However, a greater percentage of 0⁶-methylguanine (22% of the total 0⁶-methylguanine present in the DNA) compared to N-7-methylguanine (4% of the total N-7-methylguanine present in the DNA) was rendered acid soluble by S₁-nuclease. The preferential release of 0⁶-methylguanine compared to N-7-methylguanine from replicated DNA was interpreted to indicate its occurrence in local denatured regions probably generated as a result of misbase pairing.     

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.