Nuclear overhauser effect and conformational states of cellobiose in aqueous solution

A nuclear Overhauser enhancement in α-cellobiose 1-phosphate, resulting from pre-irradiation of H-1′ of the non-reducing glucosyl group, was measured and calculated theoretically. Comparison of these data reveals a complicated conformational equilibrium in aqueous solutions of the cellobiose derivative.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Summary Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 by direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec⁺ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

CD4+ lymphocytes require platelets for adhesion to immobilized fibronectin in flow: role of beta(1) (CD29)-, beta(2) (CD18)-related integrins and non-integrin receptors

The role of platelets in T-lymphocytes adhesion is not clear yet. Herpesvirus saimiri (HVS)-infected CD4(+) T-lymphocytes were placed into polystyrene plates pre-coated with fibronectin. The adherent T-cells were enumerated by image analysis. Under static condition, 38+/-10cells/mm(2) adhered and addition of gel-filtered platelets (GFP) and PMA enhanced cell adhesion 4.3- and 4.1-fold. Using PMA plus GFP 11.9-fold enhancement in cell adhesion was achieved. In contrast, under flow (200s(-1)), neither basal adhesion nor following separate addition of PMA or GFP was observed, whereas combined addition of PMA and GFP induced noticeable adhesion (34cells/mm(2)). The adhesion was inhibited by blockade of alpha(5)-integrin (CD49e, 87%), beta(2)-integrin (CD18, 78%), CD40L (60%), PSGL-1 (CD162, 60%), and CD40L plus PSGL-1 (83%). Thus, activated platelets promote activated T-cell adhesion to fibronectin under flow via integrins (alpha(5)beta(1), and alpha(L)beta(2)), CD40-CD40L and P-selectin-PSGL-1 mediated interactions.     

Glutathione propagates oxidative stress triggered by myeloperoxidase in HL-60 cells. Evidence for glutathionyl radical-induced peroxidation of phospholipids and cytotoxicity

Glutathione acts as a universal scavenger of free radicals at the expense of the formation of the glutathionyl radicals (GS*). Here we demonstrated that GS* radicals specifically interact with a reporter molecule, paramagnetic and non-fluorescent 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), and convert it into a non-paramagnetic fluorescent product, identified as 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidine (Ac-piperidine). Horseradish peroxidase-, myeloperoxidase-, and cyclooxygenasecatalyzed oxidation of phenol in the presence of H2O2 and GSH caused the generation of phenoxyl radicals and GS* radicals, of which only the latter reacted with Ac-Tempo. Oxidation of several other phenolic compounds (e.g. etoposide and tyrosine) was accompanied by the formation of GS* radicals along with a characteristic fluorescence response from Ac-Tempo. In myeloperoxidase-rich HL-60 cells treated with H2O2 and phenol, fluorescence microscopic imaging of Ac-Tempo revealed the production of GS* radicals. A thiol-blocking reagent, N-ethylmaleimide, as well as myeloperoxidase inhibitors (succinyl acetone and azide), blocked formation of fluorescent acridine-piperidine. H2O2/phenolinduced peroxidation of major classes of phospholipids in HL-60 cells was completely inhibited by Ac-Tempo, indicating that GS* radicals were responsible for phospholipid peroxidation. Thus, GSH, commonly viewed as a universal free radical scavenger and major intracellular antioxidant, acts as a pro-oxidant during myeloperoxidase-catalyzed metabolism of phenol in HL-60 cells.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Synthesis of antitumor 6-alkylidenepenicillanate sulfones and related 3-alkylidene-2-azetidinones

6-alkylidenepenicillanate sulfoxides and sulfones were synthesized on the base of 6-oxopenicillanate esters. The targeted splitting of their thiazolidine ring led to the formation of 3-alkylidene substituted 4-heteryldithio and 4-methylsulfonyl azetidin-2-ones. Some of mono and bicyclic beta-lactams revealed potent cytotoxic properties towards monolayer tumor cells in <10-microM concentrations.     

NEIL1 excises 3' end proximal oxidative DNA lesions resistant to cleavage by NTH1 and OGG1

Base excision repair is the major pathway for the repair of oxidative DNA damage in human cells that is initiated by a damage-specific DNA glycosylase. In human cells, the major DNA glycosylases for the excision of oxidative base damage are OGG1 and NTH1 that excise 8-oxoguanine and oxidative pyrimidines, respectively. We find that both enzymes have limited activity on DNA lesions located in the vicinity of the 3′ end of a DNA single-strand break, suggesting that other enzymes are involved in the processing of such lesions. In this study, we identify and characterize NEIL1 as a major DNA glycosylase that excises oxidative base damage located in close proximity to the 3′ end of a DNA single-strand break.