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Biomedical subjects

Yi Lu

Publications and source records attributed to Yi Lu.

At least 55 records · Page 3Linked to original sources
PubMed2006

A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds.

Our ability to create precise, pre-designed, spatially patterned biochemical and physical microenvironments inside polymer scaffolds could provide a powerful tool in studying progenitor cell behavior and differentiation under biomimetic, three-dimensional (3D) culture conditions. We have developed a simple and fast, layer-by-layer microstereolithography system consisting of an ultra-violet light source, a digital micro-mirror masking device, and a conventional computer projector, that allows fabrication of complex internal features along with precise spatial distribution of biological factors inside a single scaffold. Photo-crosslinkable poly(ethylene glycol) diacrylates were used as the scaffold material, and murine bone marrow-derived cells were successfully encapsulated or seeded on fibronectin-functionalized scaffolds. Fluorescently-labeled polystyrene microparticles were used to show the capability of this system to create scaffolds with complex internal architectures and spatial patterns. We demonstrate that precisely controlled pore size and shapes can be easily fabricated using a simple, computer-aided process. Our results further indicate that multi-layered scaffolds with spatially distributed factors in the same layer or across different layers can be efficiently manufactured using this technique. These microfabricated scaffolds are conducive for osteogenic differentiation of marrow-derived stem cells, as indicated by efficient matrix mineralization.

Animals↗
PubMed2006

Testing the potential for computational chemistry to quantify biophysical properties of the non-proteinaceous amino acids.

Although most proteins of most living organisms are constructed from the same set of 20 amino acids, all indications are that this standard alphabet represents a mere subset of what was available to life during early evolution. However, we currently lack an appropriate quantitative framework with which to test the qualitative hypotheses that have been offered to date as explanations for nature's "choices." Specifically, although many indices have been developed to describe the 20 standard amino acids, few or no comparable data extend to prebiotically plausible alternatives because of the costly and time-consuming bench experiments that would be required. Computational chemistry (specifically quantitative structure property relationship methods) offers a potentially fast, cost-effective remedy for this knowledge gap by predicting such molecular properties in silico. Thus, we investigated the use of various freely accessible programs to predict three key amino acid properties (hydrophobicity, charge, and size). We assessed the accuracy of these predictions by comparisons with experimentally determined counterparts for appropriate test data sets. In light of these results, and factors of software accessibility and transparency, we suggest a method for further computational assessments of prebiotically plausible amino acids. The results serve as a starting point for future quantitative analysis of amino acid alphabet evolution.

Amino Acids↗
PubMed2006

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed2006

Multi-fluorophore fluorescence resonance energy transfer for probing nucleic acids structure and folding.

Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure and dynamics of nucleic acids in solution. Such a technique often uses only one donor fluorophore and one acceptor fluorophore to probe the distance and its changes between the two labeled sites. To fully understand molecules with complicated structures, such as three- or four-way DNA junctions, several dual-fluorophore experiments have to be performed. Here, we describe an emerging alternative technique using multi-fluorophore FRET, in which simultaneous labeling of one molecule with several different fluorophores is performed to acquire all the distance information in a single experiment. This method decreases the number of experiments necessary to perform and increases the consistency of the results. In this chapter, FRET study of a tri-fluorophore-labeled DNAzyme serves as an example to illustrate the design of multi-fluorophore FRET experiments and the related data processing and analysis. The (ratio)A method used to calculate FRET efficiency in dual-fluorophore systems is extended to multi-fluorophore systems. An important difference between dual- and multi-fluorophore systems is that, when a multi-fluorophore system is used, FRET efficiency is no longer a reliable parameter to assess folding. Instead, fluorophore-to-fluorophore distance should be used.

DNA↗
PubMed2006

Fluorescent DNAzyme biosensors for metal ions based on catalytic molecular beacons.

In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for detection and quantification, making it possible to take advantage of catalytic turnovers for signal amplification. Unlike classical molecular beacons that detect only nucleic acids, catalytic molecular beacons can be applied to different DNAzymes to detect a broad range of analytes. The methods described are based on the finding that almost all known trans-cleaving DNAzymes share a similar structure comprised of a catalytic DNAzyme core flanked by two substrate recognition arms. Using a typical DNAzyme called the "8-17" DNAzyme as an example, the design of highly sensitive and selective Pb2+ sensors is described in detail. The initial design employs a single fluorophore-quencher pair in close proximity, with the fluorophore on the 5'-end of the substrate and the quencher on the 3'-end of the enzyme. Although this sensor is highly sensitive and selective at 4 degrees C, high background fluorescence is observed at higher temperatures. Therefore a new design with an additional quencher attached to the 3'-end of the substrate is employed to suppress background fluorescence. The dual quencher method allows the sensor to perform at ambient temperatures with a high signal-to-noise ratio.

Biosensing Techniques↗
PubMed2006

[Construction of recombinant plasmid pIRES2-EGFP/CCK and its expression in vivo and in vitro].

OBJECTIVE: To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK and were inserted into an eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and was observed through fluorescence microscope. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. RESULTS: A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours after the transfection. On the 4th day postinjection into the skeletal muscle of hamsters, the protein could be detected at the injection site and the fluorescence intensity became much stronger on the 14th day than that on the 4th day. On the 42nd day the protein level increased. The green fluorescence protein was never expressed in the untransfected cells. CONCLUSION: The porcine CCK gene eukaryotic expression plasmid pIRES2-EGFP/CCK is constructed successfully, and is expressed in mammal COS-7 cells and hamsters in vivo. The research paves the way for the cross immunity therapy of hamster pancreatic carcinoma.

Animals↗
PubMed2006

[Study on preparation of konjac glucomannan-hydroxypropyl methyl cellulose compression coated tablets for colonic delivery and in vitro release].

OBJECTIVE: Prepare konjac glucomannan-hydroxypropyl methyl cellulose (HPMC) compression coated tablets and study the effects of the formulation, technics and in vitro dissolution condition on drug release behavior to elevate the colon-specific effects of preparation. METHOD: Berberine hydrochloride core tablets were prepared by wet granulation technique and konjac glucomannan-HPMC mixture as the coating layer were used with compression coated technique. The effects of the formulation and technics on drug release behavior were investigated by dissolution test. The erosion of coat layer during dissolution test was investigated. RESULT: Drug almost not released in dissolution medium stimulating gastric and intestinal condition, and released completely by coating layer erosion and rupture by enzyme in stimulating colonic condition. Drug release decreased with decreasing the ratio of konjac glucomannan-HPMC and increasing coat weight (P < 0.05), compression force was not found to be a significant factor on drug release. Drug release increased with increasing the concentration of beta-mannase in dissolution medium (P < 0.05), rotation speed has no effect on drug release. The release of drug was correlative with erosion of coat layer. The mechanism of drug release were diffusion and erosion. CONCLUSION: The konjac glucomannan-HPMC compression coated tablets was a promising delivery system for drugs to be delivered to the colon.

Administration, Oral↗
PubMed2006

[Preparation and characterization of transfersomes of three drugs in vitro].

OBJECTIVE: To investigate the influence of drug properties on the encapsulation effiency (EE) and drug release of transfersomes for a proper transfersome preparation. METHOD: To prepare the transfersomes of colchicines (CLC), vincristine sulfate (VCR) and mitoxantrone hydrochloride (DHAD) with the same materials and methods, and then measure their EE. To find out the relationship between drug properties like solubility, molecular weight and charges, and EE. To performe the drug release experiments of various types of transfersomes in vitro, and compare their differences. RESULT: VCR and DHAD are lipophilic or hydrophilic, owing positive charges and large molecular weight, as a result, their EE are high, while CLC is amphipathic, neutral, and of small molecular weight, its EE is very low. As DHAD can insert into the membrane of transfersome, the drug release of DHAD-T in vitro is much slower than that of VCR-T. CONCLUSION: To prepare transfersomes with high EE, drugs that are lipophilic or hydrophilic, high molecular weight and opposite charges to the membrane should be chosen. Interaction between drugs and membrane will influnce the rate of drug release.

Antineoplastic Agents↗
PubMed2005

Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin.

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.

Animals↗
PubMed2005

Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(ii) sensing.

A Pb(ii)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(ii). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(ii)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(ii)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(ii) concentrations as low as 17 nM (3.5 ppb).

Biosensing Techniques↗
PubMed2005

Self-catalytic behavior of carbon nanotubes.

Self-catalytic behavior of multi-walled carbon nanotubes is proposed and validated experimentally under the conditions in which metal catalysis was previously figured to work only. The self-catalysis could support the nanotube nucleation and radial and axial developing processes. This result should lead to a new and better understanding of the growth mechanism of carbon nanotubes in a metal catalytic process.

Journal Article↗
PubMed2005

Blue ferrocenium azurin: an organometalloprotein with tunable redox properties.

A ferrocene derivative (2-[(methylsulfonyl)thio]ethylferrocene) (1) has been synthesized and incorporated into apo-azurin from Pseudomonas aeruginosa by covalent attachment to the highly conserved Cys112. The resulting artificial organometalloprotein (a protein containing organometallic compounds in the active site) has been characterized by UV-vis, electrospray mass spectrometry, and cyclic voltammetry (CV). Incorporation of 1 into azurin resulted in a higher solubility of the ferrocene group and improved stability of the ferrocenium species in aqueous solution, as shown by a more intense UV-vis absorption and a more reversible CV of the attached ferrocene group, respectively. The incorporation of 1 also increased the reduction potential of the complex from 402 to 579 mV (vs NHE), consistent with the ferrocene group being encapsulated inside the hydrophobic environment of the protein. Modulation of the reduction potential of ferrocene by residues near the secondary coordination sphere has also been demonstrated. Raising the pH from 4 to 9 resulted in a greater than 80 mV decrease in reduction potential of the protein-bound ferrocene (from 579 to 495 mV), while replacing Met121, an amino acid residue in close proximity to the ferrocene group with a positively charged Arg or negatively charged Glu, resulted in the predicted increase or decrease in reduction potential at all pH values. Similarly, substitution of Met121 with a more hydrophobic Leu raised the reduction potential. The increased solubility, stability, and tune-ability of this organometalloprotein make it an ideal choice for carrying out a number of biological reactions, such as long-range electron transfer or sensing. As an example of such applications, stoichiometric oxidation of ferrocytochrome c by the blue ferrocenium azurin was demonstrated.

Azurin↗
PubMed2005

Flavor characteristics of lapsang souchong and smoked lapsang souchong, a special Chinese black tea with pine smoking process.

The major volatile constituents of lapsang souchong, smoked lapsang souchong, and smoked souchong, a group of special black teas in China, were analyzed with GC and GC-MS analyses. Forty-nine constituents were identified. Longifolene and alpha-terpineol were the most abundant compounds in the aroma. Due to its special production process, the compounds longifolene, longicyclene, guaiacol, 4-methylguaiacol, 4-ethylguaiacol, etc., were identified only in this kind of black tea. The aroma constituents of tea origin decreased during the smoking process, whereas the pine terpenoids and the thermal pyrolysis products of the pine wood increased markedly. The pine material for smoking was also analyzed for the volatile constituents showing apparent selectivity of the tea leaves to absorb. There were apparent differences among the tea samples in the contents of aroma constituents. The characteristics of the aroma of the teas are discussed with their processes and the features of sensory qualities.

Camellia sinensis↗
PubMed2005

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism.

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.

Animals↗
PubMed2005

Stimuli-responsive disassembly of nanoparticle aggregates for light-up colorimetric sensing.

Controlled assembly of nanomaterials has been the focus of much research. In contrast, controlled disassembly has not received much attention, even though both processes have been shown to be important in biology. By using a Pb2+-dependent RNA-cleaving DNAzyme, we demonstrate here control of the disassembly of gold nanoparticle aggregates in response to Pb2+. In the process, we show that nanoparticle alignment plays an important role in the disassembly process, with the tail-to-tail configuration being the most optimal, probably because of the large steric hindrance of other configurations. The rate of disassembly is significantly accelerated by using small pieces of DNA to invade the cleaved substrate of the DNAzyme. Investigation of such a controlled disassembly process allows the transformation of previously designed "light-down" colorimetric Pb2+ sensors into "light-up" sensors.

Colorimetry↗
PubMed2005

Highly active and stable DNAzyme-carbon nanotube hybrids.

We report herein the generation and characterization of DNAzyme-carbon nanotube conjugates. Biotinylated DNAzyme was attached to streptavidin-functionalized MWNTs. The resultant DNAzyme-MWNT conjugates exhibited Michaelis-Menten kinetics and multiple catalytic turnovers. The specific activity of the nanoscale conjugates was approximately 1/3 of that for freely soluble DNAzyme. Such high activity may allow myriad applications ranging from the directed assembly of nanomaterials to nanoscale cellular therapeutics that rely on the RNA cleaving ability of DNAzymes.

Catalysis↗