Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity to X-irradiation in relation to cell size of CHO cells synchronized in early G1

Abstract. A population of line CHO Chinese hamster cells was synchronized by mitotic selection and allowed to enter early G₁, after which the largest and smallest cells in the population were sorted, irradiated, and their viability determined. Despite sizeable differences in volume, metabolic capability and cell cycle progression rates, an equivalent level of survival was obtained for the two populations, indicating that the factors responsible for the volume, metabolic and progression heterogeneity do not contribute greatly to radiation sensitivity.     

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Summary Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.     

Effects of exercise and conditioning on clotting and fibrinolytic activity in men

Sixty healthy men in three physical fitness categories (sedentary, on no organized fitness program; joggers, running 5-15 miles/wk; and marathoners, running greater than 50 miles/wk) were evaluated for changes in blood clotting and fibrinolytic activity before and after maximum exercise on a treadmill according to the Bruce protocol. The rate of blood clotting, as measured by prothrombin times, partial thromboplastin times and thrombin times, was accelerated by exercise (all P less than 0.005). The ability of euglobulin clots and plasma clots to lyse incorporated 125I-fibrin, termed 125I-euglobulin clot lysis (IEL) and 125I-plasma clot lysis (IPCL), were used as indexes of fibrinolytic activity. Marathoners had greater increases in fibrinolytic activity with exercise (76% compared with 63% for joggers and 55% for sedentary subjects by IEL; 427% compared with 418% for joggers and 309% for sedentary subjects by IPCL; all P less than 0.05). Fibrin degradation products increased with exercise (P less than 0.005 for the total group of 60 subjects). The absolute concentrations of alpha 2-plasmin inhibitor, alpha 2-macroglobulin, and antithrombin III increased with exercise (all P less than 0.005), but when concentrations were corrected for acute shifts of plasma water during exercise, the quantity of these inhibitors actually decreased (all P less than 0.005). The changes in clotting assays with exercise were not significantly correlated with changes in whole blood lactate, blood pyruvate, or rectal temperatures. Fibrinolytic assays before and after exercise correlated poorly to moderately with blood lactates (IEL: r = 0.441 and r = 0.425, respectively; IPCL: r = 0.294 and r = 0.544, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytological mechanisms of the development of mucosal candidiasis after exposure to a steroid aerosol preparation

Mucosal candidosis was induced in CBA mice by intraoral inoculation following treatment with corticosteroid-containing aerosol (beclomethasone dipropionate). Histologically, in hormone treated mice the adherence of the pathogen to the mucosal surface was found during the first hours after inoculation. This is followed by the formation of the germ tubes and invasion in the epithelial layer. Pseudomycelial invasion in the malpighian layer is accompanied by the leukocyte response that limits the further spread of the fungal cells. In intact mice, the inoculation is not followed by the effective attachment of the fungal cells to the mucosal surface and induction of mycotic lesions. In vitro experiments have demonstrated the enhanced adherence of fungal blastospores to the epithelial cells of the hormone treated animals, that appears to be one of the mechanisms in the pathogenesis of candidosis in these animals.     

Apoplastic Phloem Unloading in the Stem of Bean

Sucrose has been found in the apoplast of bean stems at a concentrationof 25–60 mM with an axial concentration gradient in theappropriate direction for Munch translocation. Removal of theepidermis from a 50 mm length of stem enabled the washout oflabelled photosynthate from the apoplast. The rate of labelwashout was strongly dependent on temperature, and the rateincreased on blockage of phloem pathways to the main sink forthat assimilate. Washout did not reduce when the bathed tissuewas plasmolyzed. We propose that sucrose is unloaded from thephloem into the apoplast, and a sucrose concentration is maintainedthere by a balance of sucrose uptake into sink tissue or reloadinginto the phloem. It is proposed that the apoplastic pool ofphotosynthate can act to buffer sudden changes in phloem contentswhen there are rapid changes in source-sink configuration. Key words: Sucrose, Phaseolus vulgaris, Apoplast, Phloem unloading     

Effect of prostaglandins on lipid transfer between high- and low-density lipoproteins in human plasma

Prostaglandin (PG) E1 was shown to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins to low density lipoproteins. The enhancement of the interlipoprotein lipid transfer by PGE1 was observed both at low prostaglandin concentrations under conditions of spontaneous exchange as well as in the presence of the lipoprotein-depleted plasma and the partly purified lipid transfer plasma protein. At the same time PGE2 showed no significant influence on the interlipoprotein lipid transfer. It is supposed that the effect of PGE1 is due to the PGE1-induced reorganization of the high density lipoprotein surface and that the PG-lipoprotein interaction is a factor which regulates cholesterol homeostasis.