The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.     

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.     

Growth interactions among blue-green (Anabaena Oscillarioides,Microcystis aeruginosa) and green (Chlorella sp.) algae

The growth interactions amongst the blue-green algal species Anabaena oscillarioides, Microcystis aeruginosa and the green alga, Chlorella sp. were studied both in mixed cultures and in filter cultures separated by a membrane filter in the two arms of an interaction U-tube. The role of nutrients especially phosphate upon the interaction has also been studied. Anabaena and Microcystis both inhibited the growth of Chlorella while Microcystis also inhibited the growth of Anabaena. The inhibitory effect of Microcystis was found to be dependent on high concentrations of the initial algal inocula and independent of the initial concentration of nutrients such as inorganic phosphate, indicating that the nature of the inhibition is probably due to the production of inhibitory extracellular products by Microcystis. On the other hand, the inhibitory effect of Anabaena on Chlorella is the consequence of nutrient competition with Anabaena competing more effectively for the available phosphate.     

The separation of active and inactive forms of heparin.

Heparin has been fractionated into two distinct forms. The isolation of these species was accomplished by sucrose density gradient centrifugation of heparin mixed with antithrombin-heparin cofactor. Approximately $\text{1}\text{3}$ of this mucopolysaccharide was bound to antithrombin-heparin cofactor and had potent anticoagulant activity. This component was clearly separated from the remaining $\text{2}\text{3}$ of the heparin which could not form a stable complex with antithrombin-heparin cofactor and had minimal anticoagulant activity.     

The combined effects of mixtures of ionizing radiations

Ionizing radiation is a special group of toxic agents whose general interaction can be calculated. This was demonstrated using a radiation interaction model previously published. In this paper, this model is refined and mathematically reformulated using a unified set of assumptions. It postulates the existence of a common intermediate lesion and the relative action of lesions before, at and after this common stage. General quantitative dose-effect relationships of mixed radiations can be derived from the dose-effect relationships of the components in the mixture.     

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.     

On preprocessing and antisymmetry in de novo peptide sequencing: improving efficiency and accuracy

Peptide sequencing plays a fundamental role in proteomics. Tandem mass spectrometry, being sensitive and efficient, is one of the most commonly used techniques in peptide sequencing. Many computational models and algorithms have been developed for peptide sequencing using tandem mass spectrometry. In this paper, we investigate general issues in de novo sequencing, and present results that can be used to improve current de novo sequencing algorithms. We propose a general preprocessing scheme that performs binning, pseudo-peak introduction, and noise removal, and present theoretical and experimental analyses on each of the components. Then, we study the antisymmetry problem and current assumptions related to it, and propose a more realistic way to handle the antisymmetry problem based on analysis of some datasets. We integrate our findings on preprocessing and the antisymmetry problem with some current models for peptide sequencing. Experimental results show that our findings help to improve accuracies for de novo sequencing.     

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.