Disjoining pressure and the film-height-dependent surface tension of thin liquid films: New insight from capillary wave fluctuations

In this paper we review simulation and experimental studies of thermal capillary wave fluctuations as an ideal means for probing the underlying disjoining pressure and surface tensions, and more generally, fine details of the Interfacial Hamiltonian Model. We discuss recent simulation results that reveal a film-height-dependent surface tension not accounted for in the classical Interfacial Hamiltonian Model. We show how this observation may be explained bottom-up from sound principles of statistical thermodynamics and discuss some of its implications

Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the “mass tag” (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.

CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.

Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

Diagnostic ultrasound activation of contrast agent gas bodies induces capillary rupture in mice

Interaction of diagnostic ultrasound with gas bodies produces a useful contrast effect in medical images, but the same interaction also represents a mechanism for bioeffects. Anesthetized hairless mice were scanned by using a 2.5-MHz transducer (610-ns pulses with 3.6-kHz repetition frequency and 61-Hz frame rate) after injection of Optison and Evans blue dye. Petechial hemorrhages (PHs) in intestine and abdominal muscle were counted 15 min after exposure to characterize capillary rupture, and Evans blue extravasation was evaluated in samples of muscle tissue. For 5 ml⋅kg-1 contrast agent and exposure to 10 alternating 10-s on and off periods, PH counts in muscle were approximately proportional to the square of peak negative pressure amplitude and were statistically significant above 0.64 MPa. PH counts in intestine and Evans blue extravasation into muscle tissue were significant above 1.0 MPa. The PH effect in muscle was proportional to contrast dose and was statistically significant for the lowest dose of 0.05 ml⋅kg-1. The effects decreased nearly to sham levels if the exposure was delayed 5 min. The PH effect in abdominal muscle was significant and statistically indistinguishable for uninterrupted 100-s exposure, 10-s exposure, 100 scans repeated at 1 Hz, and even for a single scan. The results confirms a previous report of PH induction by diagnostic ultrasound with contrast agent in mammalian skeletal muscle [Skyba, D. M., Price, R. J., Linka, A. Z., Skalak, T. C. & Kaul, S. (1998) Circulation 98, 290–293].

Specific accumulation of tumor-derived adhesion factor in tumor blood vessels and in capillary tube-like structures of cultured vascular endothelial cells.

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Application of capillary electrophoresis-electrospray ionization mass spectometry in the determination of molecular diversity.

By means of capillary electrophoresis coupled online to electrospray ionization MS, a library of theoretically 171 disubstituted xanthene derivatives was analyzed. The method allowed the purity and makeup of the library to be determined: 160 of the expected compounds were found to be present, and 12 side-products were also detected in the mixture. Due to the ability of capillary electrophoresis to separate analytes on the basis of charge, most of the xanthene derivatives could be resolved by simple capillary electrophoresis-MS procedures even though 124 of the 171 theoretical compounds were isobaric with at least one other molecule in the mixture. Any remaining unresolved peaks were resolved by MS/MS experiments. The method shows promise for the analysis of small combinatorial libraries with fewer than 1000 components.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

Preparation of homogeneous CNT coatings in insulating capillary tubes by an innovative electrochemically-assisted method

Preparation of homogeneous CNT coatings in insulating silica capillary tubes is carried out by an innovative electrochemically-assisted method in which the driving force for the deposition is the change in pH inside the confined space between the inner electrode and the capillary walls. This method represents a great advancement in the development of CNT coatings following a simple, cost-effective methodology.

Synthesis of Robust Hierarchical Silica Monoliths by Surface-Mediated Solution/Precipitation Reactions over Different Scales: Designing Capillary Microreactors for Environmental Applications

A synthetic procedure to prepare novel materials (surface-mediated fillings) based on robust hierarchical monoliths is reported. The methodology includes the deposition of a (micro- or mesoporous) silica thin film on the support followed by growth of a porous monolithic SiO2 structure. It has been demonstrated that this synthesis is viable for supports of different chemical nature with different inner diameters without shrinkage of the silica filling. The formation mechanism of the surface-mediated fillings is based on a solution/precipitation process and the anchoring of the silica filling to the deposited thin film. The interaction between the two SiO2 structures (monolith and thin film) depends on the porosity of the thin film and yields composite materials with different mechanical stability. By this procedure, capillary microreactors have been prepared and have been proved to be highly active and selective in the total and preferential oxidation of carbon monoxide (TOxCO and PrOxCO).

Capillary microreactors based on hierarchical SiO2 monoliths incorporating noble metal nanoparticles for the Preferential Oxidation of CO

Novel hierarchical SiO2 monolithic microreactors loaded with either Pd or Pt nanoparticles have been prepared in fused silica capillaries and tested in the Preferential Oxidation of CO (PrOx) reaction. Pd and Pt nanoparticles were prepared by the reduction by solvent method and the support used was a mesoporous SiO2 monolith prepared by a well-established sol–gel methodology. Comparison of the activity with an equivalent powder catalyst indicated that the microreactors show an enhanced catalytic behavior (both in terms of CO conversion and selectivity) due to the superior mass and heat transfer processes that take place inside the microchannel. TOF values at low CO conversions have been found to be ∼2.5 times higher in the microreactors than in the powder catalyst and the residence time seems to have a noticeable influence over the selectivity of the catalysts designed for this reaction. The Pd and Pt flexible microreactors developed in this work have proven to be effective for the CO oxidation reaction both in the presence and absence of H2, standing out as a very interesting and suitable option for the development of CO purification systems of small dimensions for portable and on-board applications.

Therapeutic drug monitoring of cefepime with micellar electrokinetic capillary chromatography: Assay improvement, quality assurance, and impact on patient drug levels

The improvement and performance of a micellar electrokinetic capillary chromatography assay for cefepime in human serum and plasma with a 50 μm id fused-silica capillary elongated from 40 to 60 cm is reported. Sample preparation with dodecylsulfate protein precipitation at pH 4.5, the pH 9.1 separation medium and the applied voltage were as reported previously[16]. The change resulted in a significant lower current, higher resolution and increased detection time intervals. The performance of the assay with multi-level internal calibration was assessed with calibration and control samples. Quality assurance data of a two year period assessed under the new conditions demonstrated the robustness of the assay. In serum samples of patients who received both cefepime and sulfamethoxazole, cefepime could not be detected due to the inseparability of the two compounds. The presence of an interference can be recognized by an increased peak width (width > 0.2 min), the appearance of a shoulder or an unresolved double peak. The patient data gathered during a three year period reveal that introduction of therapeutic drug monitoring led to a 50% reduction of the median drug level. The data suggest that therapeutic drug monitoring can help to minimize the risk of major adverse reactions and to increase drug safety on an individual basis. This article is protected by copyright. All rights reserved.