Effects of particulates and lipids on the hydraulic conductivity of Matrigel.

The hydraulic conductivity of a connective tissue is determined both by the fine ultrastructure of the extracellular matrix and the effects of larger particles in the interstitial space. In this study, we explored this relationship by examining the effects of 30- or 90-nm-diameter latex nanospheres or low-density lipoproteins (LDL) on the hydraulic conductivity of Matrigel, a basement membrane matrix. The hydraulic conductivity of Matrigel with latex nanospheres or LDL particles added at 4.8% weight fraction was measured and compared with the hydraulic conductivity of Matrigel alone. The LDL-derived lipids in the gel were visualized by transmission electron microscopy and were seen to have aggregated into particles up to 500 nm in size. The addition of these materials to the medium markedly decreased its hydraulic conductivity, with the LDL-derived lipids having a much larger effect than did the latex nanospheres. Debye-Brinkman theory was used to predict the effect of addition of particles to the hydraulic conductivity of the medium. The theoretical predictions matched well with the results from adding latex nanospheres to the medium. However, LDL decreased hydraulic conductivity much more than was predicted by the theory. The validation of the theoretical model for rigid particles embedded in extracellular matrix suggests that it could be used to make predictions about the influence of particulates (e.g., collagen, elastin, cells) on the hydraulic conductivity of the fine filamentous matrix (the proteoglycans) in connective tissues. In addition, the larger-than-predicted effects of lipidlike particles on hydraulic conductivity may magnify the pathology associated with lipid accumulation, such as in Bruch's membrane of the retina during macular degeneration and the blood vessel wall in atherosclerosis.     

Binding of streptavidin with biotinylated thermosensitive nanospheres based on poly(N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate)

Thermosensitive polymer nanospheres based on N,N-diethylacrylamide and 2-hydroxyethyl methacrylate (HEMA) have been prepared, characterized, and conjugated with biotin. The thermosensitivity of poly(N,N-diethylacrylamide) was enhanced by the incorporation of HEMA up to about 40 mol %. Atomic force microscopic images show that these particles can be closely packed even without the surface charges as in the latex particles. Biotinylation reduces the thermosensitivity of the copolymer nanospheres. The biotinylated hydrogel nanospheres showed a reduction in size upon binding with streptavidin, indicating the formation of a less hydrophilic conjugate. No aggregation of the biotinylated particles due to the cross-linking effect of streptavidin was observed. This size change could be reversed by the addition of free biotin to the system. The interaction is specific, and no such changes were observed when streptavidin was replaced by bovine serum albumin.     

Functionalized, probe-containing, latex nanospheres.

Synthesis of surface-functionalized, probe-containing latex nanospheres is described. Approximately 40,000 probe ions may be encapsulated in a nanosphere of 50 nm diameter. The probe may be a radionuclide or a lanthanide with long-lived fluorescence. Alternatively, a "cargo" of pharmaceutical interest may be used. The surface of each nanosphere contains thousands of acid groups which may be functionalized for subsequent attachment to biomolecules such as antibodies. Functionalized nanospheres have been successfully coupled to a tobacco virus.     

A third-generation H2O2 biosensor based on horseradish peroxidase-labeled Au nanoparticles self-assembled to hollow porous polymeric nanopheres

A novel third-generation biosensor for hydrogen peroxide (H2O2) was developed by self-assembling gold nanoparticles to hollow porous thiol-functionalized poly(divinylbenzene-co-acrylic acid) (DVB-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in hollow porous thiol-functionalized poly(DVB-co-AA) nanosphere latex to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups of the nanospheres. Finally, horseradish peroxidase (HRP) was immobilized on the surface of the gold nanoparticles. The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The resulting biosensor showed a wide linear range of 1.0 microM-8.0mM and a detection limit of 0.5 microM estimated at a signal-to-noise ratio of 3. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.     

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.     

黄美乳菇（红菇目,红菇科）,中国首个乳菇属变红乳菇亚属中乳汁变黄的物种

红菇属变红乳菇亚属的乳汁常变为红色,变黄的乳汁较为少见。在中国中南部亚热带壳斗科林下发现了一个乳汁迅速变黄色的物种。这是首次在中国发现的变红乳菇亚属中乳汁变黄的物种,其独特的乳汁、褐色的菌盖、稀疏的菌褶和大的具极高翼状纹饰的孢子能将其与其他已知种区分开来,在此将其描述为新种,黄美乳菇L. mirus。本研究提供了该新种的DNA条形码ITS-LSU区域和形态特征描述,并将其与其他乳汁变黄的物种如马来西亚的L. flavorosescens和L. pallidior进行了对比。这一发现为证实中国乳菇属的亚热带-热带地区与亚洲热带地区的亲缘增加了新的证据。     

Particle size and temperature effect on the physical stability of PLGA nanospheres and microspheres containing Bodipy

The purpose of this study was to investigate the effect of particle size, storage temperature, and duration of storage on the physical stability and morphology of polylactic-co-glycolic acid (PLGA) nanospheres and microspheres. PLGA nanospheres and microspheres containing the fluorescent dye, Bodipy, were prepared in varying sizes by controlling the method and degree of agitation during the emulsification phase of preparation. Mean diameters of the particles were measured by dynamic light scattering. To evaluate the effect of storage temperature and duration of storage on the extent of aggregation, nanospheres and microspheres were stored at 4°C, 25°C, 37°C, and 50°C for 6 days and then monitored using both confocal and scanning electron microscopy. The mean ±SD diameters of PLGA particles containing Bodipy were: 266.9±2.8, 351.6±1.8, 988.8±14.1, and 1865.9±67.0 nm. The extent of aggregation of the particulate delivery system decreased as the mean diameter increased, and increased as the storage temperature increased. The maximum extent of aggregation was observed with the smallest (266 nm) nanospheres. Microspheres did not aggregate. The aggregation of nanospheres was significantly reduced by introducing an additional evaporation step during preparation, suggesting that migration of residual dichloromethane from within the nanospheres may have dissolved the PLGA on the surface. The extent of aggregation of nanospheres increased as the temperature was increased from 4°C to 50°C, and decreased as particle size increased. To avoid aggregation, PLGA nanospheres should be stored at 4°C.     

Lymph node localisation of biodegradable nanospheres surface modified with poloxamer and poloxamine block co-polymers

Studies were performed to develop a sub-100 nm biodegradable colloidal system for the efficient delivery of drugs and diagnostic agents to the lymphatic system. Nanospheres of poly(lactide-co-glycolide) were prepared by interfacial polymer deposition. The nanospheres were coated with block co-polymers in order to modify their surface characteristics. Radiolabelling of the nanospheres for in vivo tracing was achieved by the incorporation of the lipophilic complex ¹¹¹In-oxine during nanosphere preparation. In vitro stability of the radiolabelled nanospheres was determined in rat serum at 37°C. The lymphatic distribution of the nanospheres was determined after subcutaneous administration to the rat. Lymphatic uptake of all coated systems was enhanced compared to the uncoated nanospheres, and a maximal uptake of 17% of the administered dose in the regional lymph nodes was achieved. These observations suggest that the nanospheres are suitable for diagnostic and therapeutic applications in clinical and experimental medicine.     

Synthesis and evaluation of uniformly sized nalidixic acid-imprinted nanospheres based on precipitation polymerization method for analytical and biomedical applications

For the first time in this work, uniform molecularly imprinted polymer (MIP) nanoparticles were prepared using nalidixic acid as a template. The MIP nanoparticles were successfully synthesized by precipitation polymerization applying methacrylic acid (MAA) as a functional monomer and trimethylolpropane trimethacrylate (TRIM) as a cross-linking monomer at different mole ratios. The morphology, binding, recognition, selectivity, and in vitro release behaviors of obtained particles were studied. The produced polymers were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetric. Furthermore, their morphology was analyzed accurately by scanning electron microscopy, photon correlation spectroscopy, and Brunauer-Emmett-Teller analysis. The nanospheres and microspheres with mean diameter values of 94 nm, 256 nm, and 1.2 μm were obtained using nalidixic acid-MAA-TRIM various mole ratios. Among the MIPs, the product with nalidixic acid-MAA-TRIM mole ratio of 1:12:12 established nanospheres with the lowest polydispersity index (0.003), an average pore diameter (12 nm), and the highest specific surface area (280 m(2) g(-1)) and selectivity factor (10.4). Results from binding experiments demonstrated that the imprinted nanospheres with a 94-nm mean diameter and a binding capacity of 28 mg of nalidixic acid per gram of polymer had higher specific affinity to nalidixic acid in contrast with the other imprinted nanospheres, microspheres, and nonimprinted particles. However, the binding performance of imprinted nanospheres in human serum was estimated using high-performance liquid chromatography analysis (binding approximately 98% of nalidixic acid). In addition, release experiments proved to be successful in the controlled release of nalidixic acid during a long period. The 20% of loaded nalidixic acid was released from the imprinted nanospheres within the first 20 h, whereas the remaining 80% was released in the after 120 h. The nalidixic acid release kinetics from the MIPs was highly affected by properties of the particles.     

Extended latex proteome analysis deciphers additional roles of the lettuce laticifer

Lettuce is an economically important leafy vegetable that accumulates a milk-like sap called latex in the laticifer. Previously, we conducted a large-scale lettuce latex proteomic analysis. However, the identified proteins were obtained only from lettuce ESTs and proteins deposited in NCBI databases. To extend the number of known latex proteins, we carried out an analysis identifying 302 additional proteins that were matched to the NCBI non-redundant protein database. Interestingly, the newly identified proteins were not recovered from lettuce EST and protein databases, indicating the usefulness of this hetero system in MudPIT analysis. Gene ontology studies revealed that the newly identified latex proteins are involved in many processes, including many metabolic pathways, binding functions, stress responses, developmental processes, protein metabolism, transport and signal transduction. Application of the non-redundant plant protein database led to the identification of an increased number of latex proteins. These newly identified latex proteins provide a rich source of information for laticifer research.     

Poly(m-phenylenediamine) nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection

In this paper, we demonstrate for the first time that poly(m-phenylenediamine) (PMPD) nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD) monomers using ammonium persulfate (APS) as an oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.     

Initial Development and Characterization of PLGA Nanospheres Containing Ropivacaine

Local anesthetics are able to induce pain relief by binding to the sodium channels of excitable membranes, blocking the influx of sodium ions and the propagation of the nervous impulse. Ropivacaine (RVC) is an amino amide, enantiomerically pure, local anesthetic largely used in surgical procedures, which present physico-chemical and therapeutic properties similar to those of bupivacaine but decreased toxicity and motor blockade. The present work focuses on the preparation and characterization of nanospheres containing RVC; 0.25% and 0.50% RVC were incorporated in poly(d,l-lactide-co-glycolide (PLGA) 50:50) nanospheres (PLGA-NS), prepared by the nanoprecipitation method. Characterization of the nanospheres was conducted through the measurement of pH, particle size, and zeta potential. The pH of the nanoparticle system with RVC was 6.58. The average diameters of the RVC-containing nanospheres was 162.7 ± 1.5 nm, and their zeta potentials were negative, with values of about ?10.81 ± 1.16 mV, which promoted good stabilization of the particles in solution. The cytotoxicity experiments show that RVC-loaded PLGA-NS generate a less toxic formulation as compared with plain RVC. Since this polymer drug-delivery system can effectively generate an even less toxic RVC formulation, this study is fundamental due to its characterization of a potentially novel pharmaceutical form for the treatment of pain with RVC.     

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.     

Molecularly imprinted micro and nanospheres for the selective recognition of 17beta-estradiol

A one-step precipitation polymerization procedure for the synthesis of molecularly imprinted polymers selective for 17beta-estradiol yielding imprinted micro and nanospheres was developed in this study and compared to templated materials obtained by conventional bulk polymerization. The polymer particles prepared by precipitation polymerization exhibited a regular spherical shape at the micro and nanoscale with a high degree of monodispersity. Moreover, the influence of the polymerization temperature, and the ratio of functional monomer to cross-linker on the size of the obtained particles was investigated. The selectivity of the imprinted micro and nanospheres was evaluated by HPLC analysis and via radioligand binding assays. HPLC separation experiments revealed that the imprinted microspheres provide higher or similar affinity to the template in contrast to imprinted polymers prepared by conventional bulk polymerization or synthesized by multi-step swelling/polymerization methods. The dimensions of the imprinted nanospheres facilitate suspension in solution rendering them ideal for binding assay applications. Results from saturation and displacement assays prove that the imprinted nanospheres exhibit superior specific affinity to the target molecule in contrast to control materials. The binding properties of the nanospheres including binding isotherms and affinity distribution were studied via Freundlich isotherm affinity distribution (FIAD) analysis. Moreover, release experiments show that 70% of rebound 17beta-estradiol was released from the imprinted nanospheres within the first 2 h, while more intimately bound 17beta-estradiol molecules (approx. 16%) were released in the following 42 h. Fitting Brunnauer-Emmet-Teller (BET) multi-point adsorption isotherms to the obtained results indicated that the micro and nanospheres are characterized by a comparatively homogenous and narrow distribution of mesopores in contrast to the corresponding bulk polymers.     

Ontogeny of anastomosed laticifers in the stem apex of <Emphasis Type="Italic">Hancornia speciosa</Emphasis> (Apocynaceae): a topographic approach

Latex is a complex plant secretion with both ecological and economic importance. There is little information currently available on the cytological aspects of the ontogenesis of anastomosed laticifers, the ducts originating through the lysis of adjacent cell walls. Hancornia speciosa is a tree typical of the Cerrado (neotropical savanna) biome. Its latex has medicinal value and is also used to produce rubber. The ontogenesis of its laticifers and the process of latex synthesis are described here. Structural, cytochemical, and ultrastructural analyses of the stem apex and phytochemical analyses of the latex were performed. Laticifer ontogenesis begins early in promeristem cells and subsequently extends through the procambial region. The laticifer precursor cells demonstrate intense metabolic activity, evidenced by starch accumulation and the proliferation of mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes—resulting in the thickening of the cell walls and accumulations of oil droplets in the cytoplasm and fibrous materials in the vacuoles. The ontogenetic process culminates with the partial dissolution of adjacent cell walls and the collapse of the cytoplasm, giving rise to anastomosed laticifers distributed throughout the phloem and adjacent regions of the cortex and medulla. The latex itself is composed of terpenes, mucilage, proteins, alkaloids, and organelle residues that form an emulsion. Laticifer development takes place in three phases: (1) the formation of the emulsion in the promeristem, (2) anastomosis and the collapse of the cytoplasm in the distal region of the procambium, and (3) the maturation of laticifers and latex storage in a central vacuole in the proximal region of the procambium.     

橡胶树凝集因子hevein基因及其启动子序列的分离与分析

橡胶树 (HeveabrasiliensisMuell._Arg .)凝集因子hevein是引起橡胶粒子凝集的主要因素 ,它是胶乳中黄色体内主要的蛋白质 ,具有几丁结合的功能。通过PCR技术扩增并克隆了橡胶树hevein基因共 6 80bp的序列。继而通过步移法分离启动子区域 130 6bp的序列 ,序列含典型的TATA盒和CAAT盒以及ABA效应元件的同源序列。为证实该基因在乳管中特异表达 ,利用Northernblot分析hevein基因在胶乳和叶片中的表达 ,同时 ,分析乙烯和ABA处理后hevein基因的表达。结果表明 ,hevein基因主要在胶乳中表达 ,乙烯和ABA对基因的表达有诱导作用。     

Anatomy of the Latex System in the Dormant Embryo of Euphorbia marginata Purch : Correlative Analysis of the Histological Organization and Topographic Distribution of Laticifers

The anatomy, morphology and topography of laticifers in themature embryo of Euphorbia marginata have been studied by conventionaland fluorescence microscopy. The mature embryo consists of awell-developed embryonic axis and two cotyledons and it containsdifferentiating vascular tissue. In the hypocotyl, six procambialstrands are present which branch and extend into the cotyledons,whereas in the root they are fused in a triarch structure. Inthe embryonic axis, the latex system consists of stelar andcortical components that are first anastomized in a cotyledonaryplexus and then are joined above in a cotyledonary system. Thestelar and cortical systems are linked by horizontal branchesto a nodal latex plexus placed under the epicotylary meristem.All the vascular laticifers, 24 in number, end unbranched inthe apex of the radicle. The cortical laticifers are profuselybranched toward the epidermis in tiny veins as far as the sub-epidermallevel. The cotyledonary laticifers follow the vascular tissueand form a network surrounding the mesophyllary areoles. Thetopography of the latex system leads one to hypothesize thatthe criteria which determine the constitution of the latex systemlareonly the essential peculiarities of idioblasts, but also correlativephenomena between growing laticifers and surrounding tissues.The stelar system appears to be under the control of vascularpolar growth, while the cortical and cotyledonary latex systemsseem to be subjected both to endogenous polar stimuli and toextra-embryonal influences. Euphorbia marginata, anatomy, embryo, laticifers     

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.     

Reversible lysozyme immobilization onto N,N′-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylenetetracarboxylic acid dimide (MPPDI) attached polymeric nanospheres

Novel hydrophobic nanospheres with an average size of 200 nm utilizing N,N′-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylenetetracarboxylic acid dimide (MPPDI) as a monomer were prepared by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA) and MPPDI conducted in an aqueous dispersion medium. The nanospheres were used for the adsorption of lysozyme. The system parameters, such as effect of the adsorption conditions (i.e. enzyme concentration, medium pH, and temperature) and the reusability of the support were studied. Specific surface area of the nonporous nanospheres was found 664 m²/g. Poly(HEMA–MPPDI) nanospheres were characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis and scanning electron microscopy (SEM). Then, poly(HEMA–MPPDI) nanospheres were used in the adsorption of lysozyme in batch system. Using an optimized adsorption protocol, 400 mg lysozyme/g nanosphere loading capacity was obtained. The adsorption phenomena appeared to follow a typical Langmuir isotherm. Lysozyme could be repeatedly adsorbed and desorbed with poly(HEMA–MPPDI) nanospheres without noticeable loss in the adsorption capacity.     

Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum

Many plants produce latex, a specialized, metabolically active cytoplasm. This is generally regarded as a defensive trait but latex may also possess additional functions. We investigated the role of latex in the dandelion species Taraxacum brevicorniculatum that contains considerable amounts of high-quality natural rubber by carrying out a comprehensive analysis of the latex proteome. We developed reliable protocols for the preparation of protein samples for one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, and subsequent mass spectrometry analysis, which led to 278 unique identifications. A gene ontology classification system based on comparisons with known Arabidopsis thaliana root proteins showed that dandelion proteins involved in lipid metabolism and transport were enriched in the latex proteome, whereas those involved in stress responses were not. We also found that proteins involved in rubber biosynthesis were distributed among different fractions of the latex proteome.