Adaptation of bulk constitutive equations to insoluble monolayer collapse at the air-water interface. (9/8297)

A constitutive equation based on stress-strain models of bulk solids was adapted to relate the surface pressure, compression rate, and temperature of an insoluble monolayer of monodendrons during collapse at the air-water interface. A power law relation between compression rate and surface pressure and an Arrhenius temperature dependence of the steady-state creep rate were observed in data from compression rate and creep experiments in the collapse region. These relations were combined into a single constitutive equation to calculate the temperature dependence of the collapse pressure with a maximum error of 5 percent for temperatures ranging from 10 degrees to 25 degrees C.  (+info)

Atomic force microscopy: a forceful way with single molecules. (10/8297)

The atomic force microscope (AFM) now routinely provides images that reveal subnanometer surface structures of biomolecules. The sensitivity and precision of AFM provide new opportunities for studying the mechanical properties of biomolecules and their interactions in their native environment.  (+info)

DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants. (11/8297)

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.  (+info)

Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. (12/8297)

Members of the BCL2 family of proteins are key regulators of programmed cell death, acting either as apoptotic agonists or antagonists. Here we describe the solution structure of BID, presenting the structure of a proapoptotic BCL2 family member. An analysis of sequence/structure of BCL2 family members allows us to define a structural superfamily, which has implications for general mechanisms for regulating proapoptotic activity. It appears two criteria must be met for proapoptotic function within the BCL2 family: targeting of molecules to intracellular membranes, and exposure of the BH3 death domain. BID's activity is regulated by a Caspase 8-mediated cleavage event, exposing the BH3 domain and significantly changing the surface charge and hydrophobicity, resulting in a change of cellular localization.  (+info)

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. (13/8297)

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.  (+info)

Altered expression profile of the surface glycopeptidolipids in drug-resistant clinical isolates of Mycobacterium avium complex. (14/8297)

Members of the Mycobacterium avium complex are the most frequently encountered opportunistic bacterial pathogens among patients in the advanced stage of AIDS. Two clinical isolates of the same strain, numbers 397 and 417, were obtained from an AIDS patient with disseminated M. avium complex infection before and after treatment with a regimen of clarithromycin and ethambutol. To identify the biochemical consequence of drug treatment, the expression and chemical composition of their major cell wall constituents, the arabinogalactan, lipoarabinomannan, and the surface glycopeptidolipids (GPL), were critically examined. Through thin layer chromatography, mass spectrometry, and chemical analysis, it was found that the GPL expression profiles differ significantly in that several apolar GPLs were overexpressed in the clinically resistant 417 isolate at the expense of the serotype 1 polar GPL, which was the single predominant band in the ethambutol-susceptible 397 isolate. Thus, instead of additional rhamnosylation on the 6-deoxytalose (6-dTal) appendage to give the serotype 1-specific disaccharide hapten, the accumulation of this nonextended apolar GPL probably provided more precursor substrate available for further nonsaccharide substitutions including a higher degree of O-methylation to give 3-O-Me-6-dTal and the unusual 4-O-sulfation on 6-dTal. Further data showed that this alteration effectively neutralized ethambutol, which is known to inhibit arabinan synthesis. Thus, in contrast with derived Emb-resistant mutants of Mycobacterium smegmatis or Mycobacterium tuberculosis, which are devoid of a surface GPL layer, the lipoarabinomannan from resistant 417 isolate grown in the presence of this drug was not apparently truncated.  (+info)

Hydrophobic hydration of amphipathic peptides. (15/8297)

Biomolecular surfaces and interfaces are commonly found with apolar character. The hydrophobic effect thus plays a crucial role in processes involving association with biomolecular surfaces in the cellular environment. By computer simulation, we compared the hydrogen bonding structures and energetics of the proximal hydration shells of the monomer and dimer from a recent study of an extrinsic membrane peptide, melittin. The two peptides were studied in their amphipathic alpha-helical forms, which possess extended hydrophobic surfaces characterized by different topography. The topography of the peptide-water interface was found to be critical in determining the enthalpic nature of hydrophobic hydration. This topographical dependence has far-reaching implications in the regulation of bioactivities in the presence of amphipathicity. This result also engenders reconsideration of the validity of using free energy parameters that depend solely on the chemical nature of constituent moieties in characterizing hydrophobic hydration of proteins and biomolecules in general.  (+info)

Stresses at the cell-to-substrate interface during locomotion of fibroblasts. (16/8297)

Recent technological improvements in the elastic substrate method make it possible to produce spatially resolved measurements of the tractions exerted by single motile cells. In this study we have applied these developments to produce maps of the tractions exerted by 3T3 fibroblasts during steady locomotion. The resulting images have a spatial resolution of approximately 5 micrometers and a maximum intensity of approximately 10(2) kdyn/cm2 (10(4) pN/micrometers2). We find that the propulsive thrust for fibroblast locomotion, approximately 0.2 dyn, is imparted to the substratum within 15 micrometers of the leading edge. These observations demonstrate that the lamellipodium of the fibroblast is able to generate intense traction stress. The cell body and posterior seem to be mechanically passive structures pulled forward entirely by this action.  (+info)