Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

A phylogenetic perspective on P transposable element evolution in Drosophila

The P element, originally described in Drosophila melanogaster, is one of the best-studied eukaryotic transposable elements. In an attempt to understand the evolutionary dynamics of the P element family, an extensive phylogenetic analysis of 239 partial P element sequences has been completed. These sequences were obtained from 40 species in the Drosophila subgenus Sophophora. The phylogeny of the P element family is examined in the context of a phylogeny of the species in which these elements are found. An interesting feature of many of the species examined is the coexistence in the same genome of P sequences belonging to two or more divergent subfamilies. In general, P elements in Drosophila have been transmitted vertically from generation to generation over evolutionary time. However, four unequivocal cases of horizontal transfer, in which the element was transferred between species, have been identified. In addition, the P element phylogeny is best explained in numerous instances by horizontal transfer at various times in the past. These observations suggest that, as with some other transposable elements, horizontal transfer may play an important role in the maintenance of P elements in natural populations.

In vitro selection for altered divalent metal specificity in the RNase P RNA

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme’s active site.

The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH1

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg−1 chlorophyll h−1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.

Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Induction of cytochrome P4501A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin or indolo(3,2-b)carbazole is associated with oxidative DNA damage.

Induction of cytochrome P4501A1 (CYP1A1) in the hepatoma Hepa1c1c7 cell line results in an elevation in the excretion rate of 8-oxoguanine (oxo8Gua), a biomarker of oxidative DNA damage and the major repair product of 8-oxo-2'-deoxyguanosine (oxo8dG) residues in DNA. Treatment of this cell line with 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), a nonmetabolized environmental contaminant, and indolo(3,2-b)carbazole (ICZ), a metabolite of a natural pesticide found in cruciferous vegetables, is shown to both induce CYP1A1 activity and elevate the excretion rate of oxo8Gua; 7,8-benzoflavone (7,8-BF or alpha-naphthoflavone), an inhibitor of CYP1A1 activity and an antagonist of the aryl hydrocarbon (Ah) receptor, reduced the excretion rate of oxo8Gua. The essential role of Ah-receptor, which mediates the induction of CYP1A1, is shown by the inability of TCDD to induce CYP1A1 and to increase excretion of oxo8Gua in Ah receptor-defective c4 mutant cells. While there was a significant 7.0-fold increase over 2 days in the excretion rate of oxo8Gua into the growth medium of TCDD-treated Hepa1c1c7 cells compared to control, no significant increase was detected in the steady-state level of oxo8dG in the DNA presumably due to efficient DNA repair. Thus, the induction of CYP1A1 appears to lead to a leak of oxygen radicals and consequent oxidative DNA damage that could lead to mutation and cancer.