In situ observation of colloidal monolayer nucleation driven by an alternating electric field.

The nucleation of crystalline materials is a hotly debated subject in the physical sciences. Despite the emergence of several theories in recent decades, much confusion still surrounds the dynamic processes of nucleation. This has been due in part to the limitations of existing experimental evidence. Charged colloidal suspensions have been used as experimental model systems for the study of crystal nucleation and structural phase transitions, as their crystallization phase diagram is analogous to that of atomic and molecular systems, but they can be visualized using microscopy. Previously, three-dimensional imaging of colloidal nucleation dynamics was achieved using confocal microscopy. However, the limited temporal resolution of the confocal microscope is of concern when trying to capture real-time colloidal crystal nucleation events. Moreover, as the thermodynamic driving force has remained undefined, data on key factors such as the critical nuclei size are at best semiquantitative. Here we present real-time direct imaging and quantitative measurements of the pre- and post-nucleation processes of colloidal spheres, and the kinetics of nucleation driven by an alternating electric field, under well-defined thermodynamic driving forces. Our imaging approach could facilitate the observation of other rarely observed phenomena, such as defect and grain-boundary formation and the effects of foreign particles during crystallization. Furthermore, it may prove useful in identifying optical and biological technologies based on colloids.

Controlled formation of colloidal structures by an alternating electric field and its mechanisms.

A detailed phase diagram, revealing a variety of processes including various colloidal structures of monodisperse charged colloidal particles from the colloidal chains, vortex rings, three-dimensional aggregation to a two-dimensional crystal under different frequencies, and strengths of an alternating electric field, is obtained for the first time. The occurrence of different colloidal structures is driven by the electrohydrodynamic interaction and induced dipolar interaction near the polarized layer on the electrode. This simple colloidal system can be employed as a model system to understand the complex phase behavior of the assembly/aggregation of the nanoparticles and biomacromolecules under external perturbation. Detailed phase diagram provides vital guidance for the fabrication of desired colloidal structures with single-particle resolution, which could be employed as a sort of templates for nanolithography or imprinting. Moreover, the sensitivity of the electrohydrodynamic interaction on the particle size and the dependence of the convective flow on the frequency and strength could be utilized in microfluidic devices for manipulating nanoparticles, biomacromolecules, and vesicles.

Aggregation of antifreeze protein and impact on antifreeze activity.

Antifreeze protein type III aggregates once the concentration exceeds a critical value, the so-called critical aggregation concentration (CAC). It was found for the first time that the aggregation of antifreeze protein exerts a direct impact on the antifreeze efficiency. It follows from our measurements that the AFP III above CAC will enhance the antifreeze activity because of the increase of the kink kinetics barrier of surface integration. This is attributed to the optimal packing of AFP III molecules on the surface of the ice nucleus as well as ice crystals above CAC. This study will extend our understanding of the antifreeze mechanism of antifreeze protein monomers as well as antifreeze aggregates on ice nucleation and shed light on the selection of antifreeze agents.

Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis.

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln(58) had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.

Identification of peptides that bind to irradiated pancreatic tumor cells.

PURPOSE: Peptides targeting tumor vascular cells or tumor cells themselves have the potential to be used as vectors for delivering either DNA in gene therapy or antitumor agents in chemotherapy. We wished to determine if peptides identified by phage display could be used to target irradiated pancreatic cancer cells. METHODS AND MATERIALS: Irradiated Capan-2 cells were incubated with 5 x 10(12) plaque-forming units of a phage display library. Internalized phage were recovered and absorbed against unirradiated cells. After five such cycles of enrichment, the recovered phage were subjected to DNA sequencing analysis and synthetic peptides made. The binding of both phage and synthetic peptides was evaluated by fluorescence staining and flow cytometry in vitro and in vivo. RESULTS: We identified one 12-mer peptide (PA1) that binds to irradiated Capan-2 pancreatic adenocarcinoma cells but not to unirradiated cells. The binding of peptide was significant after 48 h incubation with cells. In vivo experiments with Capan-2 xenografts in nude mice demonstrated that these small peptides are able to penetrate tumor tissue after intravenous injections and bind specifically to irradiated tumor cells. CONCLUSION: These data suggest that peptides can be identified that target tumors with radiation-induced cell markers and may be clinically useful.

Gelation with Small Molecules: from Formation Mechanism to NanostructureArchitecture.

The mechanism of fiber and fiber network formation of small molecular gelling agents is treated on the basis of a generic heterogeneous nucleation model. The formation of a crystallite fiber network can take place via the so-called crystallographic mismatch branching. At very low supersaturations, unbranched fibers form predominantly. As supersaturation increases, small-angle crystallographic mismatch branching occurs at the side face of growth fibers. At very high supersaturations, the so-called wide-angle crystallographic mismatch branching becomes kinetically favorable. Both give rise to the formation of fiber networks, but of different types. Controlling the branching of the nanofibers of small molecular gelatins allows us to achieve the micro/nanostructure architecture of networks having the desired rheological properties. In this regard, the engineering of supramolecular functional materials can be achieved by constructing and manipulating the micro/nanostructure in terms of a "branching creator", or by tuning processing conditions.

Novel bis-acac-O,O-Ir(III) catalyst for anti-Markovnikov, hydroarylation of olefins operates by arene CH activation.

A novel, thermally stable, homogeneous Ir catalyst for the anti-Markovnikov, hydroarylation of olefins is shown to operate by arene CH activation via the formation of a bisacac-O,O phenyl-Ir(III) species.

Rhodium complexes bearing tetradentate diamine-bis(phenolate) ligands.

Using tetradentate, dianionic ligands, several new rhodium complexes have been prepared. Some of these diamine-bis(phenolate) compounds, are active for C-H activation of benzene. These complexes are air and thermally stable. All four complexes were characterized by X-ray diffraction.

Biocompatibility investigation of polyethylene glycol and alginate-poly-L-lysine for islet encapsulation.

The use of microencapsulation with alginate-poly-l-lysine (PLL) as the encapsulation material has been hampered by overgrowth of collagen around implanted capsules. Studies have shown that poly(ethylene glycol) (PEG) has higher biocompatibility than PLL. In this project, we examined the biocompatibility of PEG in comparison with PLL in the Lewis rat model. Capsules made from either PEG or PLL were implanted into Lewis rats in three anatomical sites: subcutaneous (SC), intramuscular (IM), and intra-epididymis (IE). After 2 or 4 weeks, capsules were retrieved, sectioned, and stained with Sirius Red for analysis of fibrotic overgrowth with ImageJ software. The results were statistically analyzed using either unpaired t test or analysis of variance (ANOVA). PEG demonstrated significantly better biocompatibility in SC, at both 2 and 4 weeks, and IE at 2 weeks (p < 0.0001). No significant differences were found in IM implantation at either time point (p = 0.36) between the two materials. However, there was significantly heavier fibrotic overgrowth around PEG capsules in IE than PLL capsules at 4 weeks (p < 0.01). When compared among the anatomical sites, IM implantation demonstrated significantly less fibrotic overgrowth than other sites for both materials (p < 0.01). In conclusion, PLL and PEG may induce different levels of fibrosis based on anatomical location and duration of implantation.

Determination of elastic constants of two-dimensional close-packed colloidal crystals.

We present a noncontact, accurate, and efficient methodology for determination of elastic constants in two-dimensional colloidal crystals via the calculation of the local strain fluctuation of particles. The hexagonally close-packed colloidal crystals form from microsized particles subjected to an alternating electric field. The elastic constants in the thermodynamic limit are obtained by the extrapolation of finite-size scaling of the elastic moduli as the functions of the frequency and field strength. It is found that the elastic constants in our system are larger than those in non-close-packed colloidal crystals reported before. This technique could be a rational method to study the elasticity of soft solids.

The granular chloride channel ClC-3 is permissive for insulin secretion.

Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.

Two scenarios for colloidal phase transitions.

A two-dimensional assembly of charged colloidal particles induced by an alternating electric field was studied in real space by means of digital video microscopy. Phase transitions occur from a highly ordered colloidal monolayer to an isotropic suspension by changing the field strength or frequency (in the appropriate range). In particular, it is found that the strength-dependent phase transition is an infinite-order phase transition, in contrast with the frequency-dependent phase transition, which is a second-order phase transition.

Templating and supersaturation-driven anti-templating: principles of biomineral architecture.

The structural synergy between biominerals (CaCO(3), hydroxyapatite) and biosubstrates were examined for the first time. The templating effect of substrate and a newly identified supersaturation-driven interfacial structure mismatch effect were identified in the context of a new nucleation model. It follows that the heterogeneous nucleation which corresponds to a good structural match and synergy between biominerals and substrates will promote an ordered, compact, and tough complex biomineral structure, and occur only at low supersaturations, whereas at high supersaturations the heterogeneous nucleation associated with a poor structural match and synergy between biominerals and substrates will become dominant due to supersaturation-driven interfacial structural mismatch. The latter normally results in a disordered and porous structure. A phenomenon, so-called microgravity-driven homogeneous nucleation, was also examined. It turns out that microgravity will suppress convection and consequently promote homogeneous-like nucleation during biomineralization. This could be responsible for microgravity-induced osteoporousis.

Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function.

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1-6 and TMDs 7-12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204-214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFC(D215-R263 Delta), hRFC(K204-R263 Delta) and hRFC(K204-R214 Delta) proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6 S )5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFC(K204-R263 Delta) restored low levels of transport (16-21% of the wild type). Insertion of the ThTr1 linkers into hRFC(D215-R263 Delta) at position 215 restored 60-80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein.

The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time.

Mitotic arrest induced by XK469, a novel antitumor agent, is correlated with the inhibition of cyclin B1 ubiquitination.

XK469 (NSC 697887) is a novel antitumor agent with broad activity against a variety of tumors. Previous studies suggest that XK469 is a topoisomerase II beta poison with functional activity similar to that of 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The goal of our study was to investigate its mechanism of action further using a human HCT-116 (H116) colon tumor cell model. Concentration-survival curves with continuous exposure indicated that XK469 had low cytotoxic activity against H116 cells. Cell cycle analysis revealed that XK469 is a phase-specific cell cycle blocker that is associated with increased levels of cyclin B1, cyclin A and p53 but not CDK1 (cdc2) or cyclin E. In contrast, treatment of H116 cells with m-AMSA caused a total degradation of both cyclin A and B1 but enhanced expression of cyclin E and p53. Accumulation of cyclin B1 in XK469-treated cells was correlated with the inhibition of cyclin B1 ubiquitination, a metabolic process mandatory for proteasome-mediated protein turnover. However, no inhibition of cyclin B1 ubiquitination was detected in cells treated with m-AMSA or colchicine, a known mitotic inhibitor. Furthermore, unlike m-AMSA, XK469 did not induce caspase activation or apoptotic cell death in H116 cells. Our results suggest that XK469 is a phase-specific cell cycle inhibitor with a unique mechanism of action that is correlated with the inhibition of cyclin B1 ubiquitination and its accumulation at early M phase.