A binding mode of lambda-[tris(1,10-phenanthroline)ruthenium(II)]2+ exhibiting preference for purine-3',5'-pyrimidine sites of DNA.

Molecular mechanics calculations and molecular dynamics simulations have been used to study the binding of the partially inserted major groove complex of Lambda-[Ru(1,10-phenanthroline)3]2+ with DNA. Energy refinements of this complex showed a clear preference for binding at purine-3',5'-pyrimidine sites over pyrimidine-3',5'-purine sites. The basis for this difference is shown to be a slight change in the binding orientation induced by interchanging the purine and pyrimidine bases. This in turn provides for a better secondary interaction with the helix backbone at a point beyond the immediate binding site. It is this secondary interaction that provides the additional energetic stabilisation for complexes formed at purine-3',5'-pyrimidine sites. Molecular dynamics simulations including explicit representation of solvent support these conclusions and provide an insight into the positional stability of the ligand at a particular site. Repuckering of specific deoxyribose rings to the C3'-endo conformation seems to be an important feature of the DNA/ligand complex.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA     

ESR studies on the orientation of cholesteryl ester in phosphatidylcholine multilayers

The alignment of cholesteryl esters in multilayer phosphatidylcholine membranes was investigated using two spin-labelled cholesteryl esters: 10 : 3 ester ($\text{I}$) and 1 : 14 ester ($\text{II}$). The nitroxide label of $\text{I}$ is aligned in the membrane with a very large angle of tilt (47° ± 1.5°) with respect to the normal to the membrane surface; $\text{II}$ does not show such a tilt. $\text{I}$ gives spectra corresponding to immobilized label while $\text{II}$ gives nearly isotropic spectra. Ascorbate treatment of the multilayers shows that the labels in $\text{I}$ and $\text{II}$ are not present at the phosphatidylcholine-water interphase.The data supports a ‘horseshoe’ configuration for the cholesteryl ester in the bilayer, with both the fatty acid chain and the cholesteryl moiety extending deep into the hydrophobic region of the membrane and with the ester linkage near the surface.     

An extended RNA binding site for the yeast branch point-binding protein and the role of its zinc knuckle domains in RNA binding

The highly conserved branch point sequence (BPS) of UACUAAC in Saccharomyces cerevisiae is initially recognized by the branch point-binding protein (BBP). Using systematic evolution of ligands by exponential enrichment we have determined that yeast BBP binds the branch point sequence UACUAAC with highest affinity and prefers an additional adenosine downstream of the BPS. Furthermore, we also found that a stem-loop upstream of the BPS enhances binding both to an artificially designed RNA (30-fold effect) and to an RNA from a yeast intron (3-fold effect). The zinc knuckles of BBP are partially responsible for the enhanced binding to the stem-loop but do not appear to have a significant role in the binding of BBP to single-strand RNA substrates. C-terminal deletions of BBP reveal that the linker regions between the two zinc knuckles and between the N-terminal RNA binding domains (KH and QUA2 domains) and the first zinc knuckle are important for binding to RNA. The lack of involvement of the second highly conserved zinc knuckle in RNA binding suggests that this zinc knuckle plays a different role in RNA processing than enhancing the binding of BBP to the BPS.     

Renal autoregulation: new perspectives regarding the protective and regulatory roles of the underlying mechanisms

When the kidney is subjected to acute increases in blood pressure (BP), renal blood flow (RBF) and glomerular filtration rate (GFR) are observed to remain relatively constant. Two mechanisms, tubuloglomerular feedback (TGF) and the myogenic response, are thought to act in concert to achieve a precise moment-by-moment regulation of GFR and distal salt delivery. The current view is that this mechanism insulates renal excretory function from fluctuations in BP. Indeed, the concept that renal autoregulation is necessary for normal renal function and volume homeostasis has long been a cornerstone of renal physiology. This article presents a very different view, at least regarding the myogenic component of this response. We suggest that its primary purpose is to protect the kidney against the damaging effects of hypertension. The arguments advanced take into consideration the unique properties of the afferent arteriolar myogenic response that allow it to protect against the oscillating systolic pressure and the accruing evidence that when this response is impaired, the primary consequence is not a disturbed volume homeostasis but rather an increased susceptibility to hypertensive injury. It is suggested that redundant and compensatory mechanisms achieve volume regulation, despite considerable fluctuations in distal delivery, and the assumed moment-by-moment regulation of renal hemodynamics is questioned. Evidence is presented suggesting that additional mechanisms exist to maintain ambient levels of RBF and GFR within normal range, despite chronic alterations in BP and severely impaired acute responses to pressure. Finally, the implications of this new perspective on the divergent roles of the myogenic response to pressure vs. the TGF response to changes in distal delivery are considered, and it is proposed that in addition to TGF-induced vasoconstriction, vasodepressor responses to reduced distal delivery may play a critical role in modulating afferent arteriolar reactivity to integrate the regulatory and protective functions of the renal microvasculature.     

Bending rigidities of cell surface molecules P-selectin and PSGL-1

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100 pN nm².     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism

Linear dichroism (LD) can be used to study the alignment of absorbing chromophores within long molecules. In particular, Couette flow LD has been used to good effect in probing ligand binding to DNA and to fibrous proteins. This technique has been previously limited by large sample requirements. Here we report the design and application of a new micro-volume Couette flow cell that significantly enhances the potential applications of flow LD spectroscopy by reducing the sample requirements for flow linear dichroism to 25 microL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is approximately 1). The micro-volume Couette cell has also enabled the measurement of fluorescence-detected Couette flow linear dichroism. This new technique enables the orientation of fluorescent ligands to be probed even when their electronic transitions overlap with those of the macromolecule and conversely. The potential of flow-oriented fluorescence dichroism and application of the micro-volume Couette LD cell are illustrated by the collection of data for DNA with minor groove and intercalating ligands: DAPI, Hoechst, and ethidium bromide. As with conventional fluorescence, improved sensitivity compared with absorbance LD is to be expected after instrumentation optimization.     

Dimeric galectin-1 induces surface exposure of phosphatidylserine and phagocytic recognition of leukocytes without inducing apoptosis

We report that human galectin-1 (dGal-1), a small dimeric beta-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface beta-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis.