Biomedical subjects

Ming Lei

Publications and source records attributed to Ming Lei.

At least 55 records · Page 3Linked to original sources

PubMed2004

Hard and soft micromachining for BioMEMS: review of techniques and examples of applications in microfluidics and drug delivery.

Recent development in microfabrication (micromachining, microelectromechanical systems, MEMS) permits the integration of hard and soft structures, and enables the design of controllable microfluidic systems, which may be applied to drug delivery. In this paper, we present a tutorial review of both classical "hard" and more recent "soft" micromachining techniques. We then provide examples where these techniques are combined to produce hydrogel-based microfluidic control systems. The most complex of these systems utilizes a very small hydrogel based on phenylboronic acid to control the flow of an insulin solution in response to changes in glucose concentration.

Drug Delivery Systems↗

PubMed2004

Intrinsic versus mutation dependent instability/flexibility: a comparative analysis of the structure and dynamics of wild-type transthyretin and its pathogenic variants.

Transthyretin (TTR) is one of the about 20 known human proteins associated with amyloidosis which is characterized by the accumulation of amyloid fibrils in tissues or extracellular matrix surrounding vital organs. Unlike Alzheimer's fibrils that comprise a fragment of a large precursor protein, TTR amyloid fibrils are composed of both full-length protein and fragments of the molecule. The native state of TTR is a homotetramer with eight beta-strands organized into a beta-sandwich in each monomer. To elucidate the structural reorganization mechanisms preceding amyloid formation, it is important to characterize the dynamic features of the wild-type native state as well as to reveal the influence of disease-associated mutations on the structure and dynamics. Molecular dynamics (MD) simulations complement X-ray crystallography and D-H exchange to capture the intrinsically unstable/flexible sites of the wild-type as well as the mutation dependent unstable sites of the pathogenic variants. Our results of MD simulations have shown that the Leu55-->Pro (L55P) mutation occurs in an intrinsically unstable site, leading to substantial local and global structural changes. This observation supports the early speculation that the C-strand-loop-D-strand rearrangement leads to the formation of amyloidogenic intermediates. In addition to the D strand, the alpha-helical region and the strands at the monomer-monomer interface are also intrinsically unstable. The central channel of L55P-TTR undergoes opening and closing fluctuations, which may provide an explanation for the fact that while the mutation is far from the channel, the mutant shows a substantial low binding affinity of thyroxine.

Binding Sites↗

PubMed2004

Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts.

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.

Biological Factors↗

PubMed2004

[Analysis of acute respiratory dysfunction as a risk factor of multiple injuries].

OBJECTIVE: To study the relationship between multiple injuries and acute respiratory dysfunction (ARD). METHODS: One hundred and ten patients with multiple injuries admitted during the last 6 years were retrospectively analyzed. With ARD as the dependent variable and other 11 factors as independent variables, all analysis was done statistically on computer to identify the complication of ARD as a risk factor. RESULTS: Multivariable and single argument analysis showed systemic inflammatory response syndrome (SIRS), pulmonary contusion, co-existing lung disease as chronic obstructive pulmonary disease (COPD) and pneumonia, moderate hemothorax and pneumothorax, duration of shock over 12 hours, age over 55 years, injury severity score(ISS)>24 were high risk factors. CONCLUSION: Patients with multiple injuries with high risk factors should be kept under closer observation in order to prevent ARD and multiple organ dysfunction syndromes (MODS).

Adolescent↗

PubMed2003

Characteristics and mechanisms of the two types of photoelectric differential response of bacteriorhodopsin-based photocell.

Bacteriorhodopsin (BR)-based photocells have been assigned possessing differential photoelectric response. But recently we found that the differential response described before, which occurred in milliseconds to seconds, outputting a positive pulse when light was on and a negative pulse when light was off, was not the intrinsic property of the BR molecule. It was partially caused by the measuring circuit. By measuring the photoelectric response signal of the BR film photocell to a short laser pulse, the impulse response function of the BR film photocell was obtained by data fitting with MATLAB software. A simulation system was accordingly developed. The output response signals of the BR film photocell under different stepping incident light were calculated. By simulation and analysis, it was concluded that the differential response caused by the intrinsic property of the BR molecule happened in microseconds time scale, and it produced a negative pulse when light was on and a positive pulse when light was off. It was much faster but much weaker than that described before.

Bacteriorhodopsins↗

PubMed2003

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA.

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.

Base Sequence↗

PubMed2003

Inhibition of p38 MAPK decreases myocardial TNF-alpha expression and improves myocardial function and survival in endotoxemia.

OBJECTIVES: The role of p38 mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide (LPS)-induced myocardial dysfunction has not been clearly defined. Our aim was to investigate the contribution of p38 MAPK in myocardial tumor necrosis factor-alpha (TNF-alpha) expression, cardiac function and survival during acute endotoxemia in mice. METHODS: Acute endotoxemia was induced by LPS (10 mg/kg, i.p.) in mice. Two hours after LPS treatment, left ventricular (LV) function was assessed. Phosphorylation of p38 MAPK was measured by Western blotting. TNF-alpha mRNA and protein levels were determined by semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: LPS rapidly increased phosphorylation of p38 MAPK, followed by TNF-alpha mRNA expression and protein expression in the LV myocardium. Pre-treatment of the p38 MAPK inhibitor SB202190 (2 mg/kg, i.p.) decreased TNF-alpha mRNA and protein by 65 and 36%, respectively (P<0.05). Immunohistochemical staining confirmed that cardiomyocytes were the major source of TNF-alpha production in the myocardium and blocking p38 MAPK activation inhibited TNF-alpha expression in response to LPS. Pre-treatment of SB202190 or a TNF-alpha antagonist etanercept (2 mg/kg, i.p) significantly reversed LPS-induced LV depression (P<0.05). LPS (20 mg/kg, i.p.) induced 94% mortality in mice within 72 h and pre-treatment with SB202190 and etanercept decreased LPS-induced mortality to 65 and 40%, respectively (P<0.01). CONCLUSION: p38 MAPK activation represents an important mechanism leading to myocardial TNF-alpha production and cardiac dysfunction during acute endotoxemia in mice. Our data suggest that p38 MAPK is a potential therapeutic target of endotoxemia.

Acute Disease↗

PubMed2003

Two bipartite NLSs mediate constitutive nuclear localization of Mcm10.

Some components of the pre-replicative complex (pre-RC) are in the nucleus only during the G1 phase, while others are constitutively nuclear throughout the cell cycle. A nuclear localization signal (NLS) mediating the G1-specific nuclear localization has been characterized. We report here the identification of bipartite NLSs in constitutively nuclear pre-RC components, including the origin recognition complex, Mcm10 and Noc3. Interestingly, in a subgroup of these pre-RC components, two putative NLSs were found in each protein. To examine whether multiple NLSs are required for these proteins to be nuclear throughout the cell cycle, we characterized the two NLSs in Mcm10. We show that at least one of the two NLSs is required for directing Mcm10 into the nucleus and that either of them is sufficient to bring the green fluorescence protein into the nucleus. Thus, Mcm10 contains two functional NLSs and either is sufficient for the constitutive nuclear localization of Mcm10.

Blotting, Western↗

PubMed2003

A novel zinc finger is required for Mcm10 homocomplex assembly.

Mcm10 is a DNA replication factor that interacts with multiple subunits of the MCM2-7 hexameric complex. We report here that Mcm10 self-interacts and assembles into large homocomplexes (approximately 800 kDa). A conserved domain of 210 amino acid residues is sufficient for mediating self-interaction and complex assembly. A novel zinc finger within the conserved domain, CX10CX11CX2H, is essential for the homocomplex formation. Mutant alleles with amino acid substitutions at conserved cysteines and histidine in the zinc finger fail to assemble homocomplexes. A defect in homocomplex assembly correlates with defects in DNA replication and cell growth in the mutants. These observations suggest that homocomplex assembly is essential for Mcm10 function. Multisubunit Mcm10 homocomplexes may provide the structural basis for Mcm10 to interact with multiple subunits of the MCM2-7 hexamer.

Alleles↗

PubMed2003

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

Amino Acid Sequence↗

PubMed2003

High resolution CryoFESEM of microbial surfaces.

The outer surfaces of three microorganisms, Giardia lamblia, Enterococcus faecalis, and Proteus mirabilis, were investigated by cryo-immobilization followed by sublimation of extracellular ice and cryocoating with either Pt alone or Pt plus carbon. Cryocoated samples were examined at -125 degrees C in either an in-lens field emission SEM or a below-the-lens field emission SEM. Cryocoating with Pt alone was sufficient for low magnification observation, but attempts to do high-resolution imaging resulted in radiolysis and cracking of the specimen surface. Double coating with Pt and carbon, in combination with high resolution backscatter electron detectors, enabled high-resolution imaging of the glycocalyx of bacteria, revealing a sponge-like network over the surface. High resolution examination of bacterial flagella also revealed a periodic substructure. Common artifacts included radiolysis leading to "cracking" of the surface, and insufficient deposition of Pt resulting in the absence of detectable surface topography.

Animals↗

PubMed2003

Laser-light scattering, a new method for continuous monitoring of platelet activation in circulating fluid.

We evaluated a novel technique of laser-light scattering (LLS) to detect platelet-volume changes continuously, reflecting platelet aggregation in circulating fluid. Carotid arteries from 20 dogs were mounted in a dual perfusion chamber. Balloon angioplasty (BA) was performed and arteries perfused with platelet-rich plasma (PRP). A He-Ne laser beam was passed through cuvettes connected to tubing draining the arteries. From the angle of incidence, the average volume of aggregates was measured by the ratio of scattering light at 1 to 5 degrees' spread on the diode array of a multichannel analyzer. Platelet volume varied linearly with the scattered light ratio at 1 to 5 degrees (y = -24.2 + 27.6 x [y = particle size, microm(3); x = scattered light ratio at 1/5 degrees]). For comparison, we used an electronic particle counter (Coulter counter) to measure platelet volume. P-selectin expression was measured to confirm platelet activation. Comparing 10 uninjured and 10 BA-injured arteries, we found that platelet volume as measured with LLS increased from 21.6 +/- 4.1 to 52.1 +/- 12.5 microm(3) (P < .003); as measured with the Coulter counter, it increased from 29.9 +/- 2.4 to 62.3 +/- 7.0 microm(3) (P < .005). Six BA-injured arteries perfused with PRP and aspirin (0.2 mg/mL) were compared with six arteries treated with BA alone. The aspirin decreased platelet volume as measured with LLS from 56.2 +/- 11.8 to 40.2 +/- 12.7 microm(3) (P < .01); the Coulter counter revealed a decrease from 51.9 +/- 18.5 to 38.8 +/- 14.2 microm(3) (P < .001). Coulter counter and LLS results were correlated: r = 0.74, P < .05. The peak of P-selectin expression coincided with peak platelet volume. These data demonstrate that increases in circulating-platelet size stimulated by endovascular injury can be reliably and continuously monitored with the use of LLS.

Angioplasty, Balloon↗

PubMed2003

Why is Leu55-->Pro55 transthyretin variant the most amyloidogenic: insights from molecular dynamics simulations of transthyretin monomers.

Transthyretin (TTR) is one of the known human amyloidogenic proteins. Its native state is a homotetramer with each monomer having a beta-sandwich structure. Strong experimental evidence suggests that TTR dissociates into monomeric intermediates and that the monomers subsequently self-assemble to form amyloid deposits and insoluble fibrils. However, details on the early steps along the pathway of TTR amyloid formation are unclear, although various experimental approaches with resolutions at the molecular or residue level have provided some clues. It is highly likely that the stability and flexibility of monomeric TTR play crucial roles in the early steps of amyloid formation; thereby, it is essential to characterize initial conformational changes of TTR monomers. In this article we probe the possibility that the differences in the monomeric forms of wild-type (WT) TTR and its variants are responsible for differential amyloidogenesis. We begin with the simulations of WT, Val30-->Met (V30M), and Leu55-->Pro (L55P) TTR monomers. Nanosecond time scale molecular dynamics simulations at 300 K were performed using AMBER. The results indicate that the L55P-TTR monomer undergoes substantial structural changes relative to fluctuations observed in the WT and V30M TTR monomers. The observation supports earlier speculation that the L55P mutation may lead to disruption of the beta-sheet structure through the disorder of the "edge strands" that might facilitate amyloidogenesis.

Amyloid↗

PubMed2003

[Holographic recording properties of BR-D96N film].

BR-D96N was a genetically mutated product of bacteriorhodopsin (BR) with obvious improved photochromic effect. Compared with the wild type BR, BR-D96N had a lifetime of M state prolonged to 5 min, showing obvious saturation absorption and lower light intensity in saturation absorption (0.4 mW/cm(2)). In case of holographic recording, dynamic grating was recorded in the BR-D96N film, its characteristic parameter was not light exposure energy but light intensity. The writing time of the holographic grating depended on the recording light intensity. The higher the recording light intensity, the faster the grating builds up. Under a weak reconstruction light, the recording light intensity resulting in maximal diffraction efficiency was consistent with the saturation absorption intensity. The reconstruction light could partly erase the grating. With lower intensity of reconstruction light, higher diffraction efficiency (1.8%) could be reached, but the diffraction intensity was not high. To get highest diffraction intensity, a properly high intensity of reconstruction light was needed (80 microW/cm(2)). The result of these experiments showed that holographic images could be recorded on the BR-D96N film.

Bacteriorhodopsins↗

PubMed2003

[Dynamic changes of platelet number in early multiple injury and it is effect on prognosis].

OBJECTIVE: To study the dynamic changes of platelet number in early multiple injured patients and its clinical significance. METHODS: Ninety patients with multiple injury less than 48 hours were selected. Blood analysis was dynamicly investigated at 1, 24, 72, 120 and 168 hours after in Hospital, and acute physiology and chronic health evaluation III (APACHE III) and injury seriousness scale (ISS) scores were recorded. RESULTS: The platelet number declined after injury, reached the lowest point at 72 hours after in hospital, then restored to the normal level at 120 hours. In the platelet decrease group, the trauma was more critically[ISS (29.60+/-9.80) scores vs. (22.30+/-12.00) scores, (P<0.05)] and the incidence of multiple organs dysfunction syndrome (MODS) was higher than normal group (83.9% vs. 62.7%, P<0.05). In the death group, the number of platelet was declined[ (30.1+/-10.3) hours vs. (51.3+/-14.8) hours, (P<0.05) and the lasting tine was longer[ (63.7+/-11.0) hours vs. (54.2+/-13.4) hours, (P<0.05). CONCLUSION: The platelet number declines after multiple injury. The decrease and continuously lower level of platelet suggest poor prognosis.

Adolescent↗

PubMed2002

Endothelial nitric-oxide synthase enhances lipopolysaccharide-stimulated tumor necrosis factor-alpha expression via cAMP-mediated p38 MAPK pathway in cardiomyocytes.

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-alpha expression in neonatal mouse cardiomyocytes. TNF-alpha expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-alpha expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-alpha expression in cardiomyocytes. NOS inhibitor l-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes, whereas NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 microm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS(-/-) cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS(-/-) cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS(-/-) cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-alpha expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-alpha expression. Moreover, the effects of NO on LPS-stimulated TNF-alpha expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.

Animals↗

PubMed2002

Gains in sensitivity with a device that mixes microarray hybridization solution in a 25-microm-thick chamber.

A microarray hybridization system that allows mixing in volumes comparable to those used by glass coverslips is presented. This system is composed of a disposable flexible lid that binds to 1 in. x 3 in. glass slides via an adhesive gasket, forming a uniform 25-microm-thick hybridization chamber. This chamber rests on a base unit for temperature control. The lid contains two air-driven bladders that continuously mix the hybridization fluid. Mixing enhances sensitivity from a typical microarray experiment 2-3-fold. Mixing is particularly effective at high spotted probe and low labeled target concentrations and overcoming local target depletion that occurs when homologous probes are spotted in close proximity. Mixing appears to be compatible with most hybridization conditions; however, mix versus no-mix control experiments should be performed. Also covered are a number of microfluidic issues related to manufacturing, filling, mixing, and packaging.

Bacteriophage lambda↗

PubMed2002

Cooperative binding of single-stranded telomeric DNA by the Pot1 protein of Schizosaccharomyces pombe.

The fission yeast Pot1 (protection of telomeres) protein is a single-stranded telomeric DNA-binding protein and is required to protect the ends of chromosomes. Its N-terminal DNA-binding domain, Pot1pN, shows sequence similarity to the first OB fold of the telomere-binding protein alpha subunit of Oxytricha nova. The minimal-length telomeric ssDNA required to bind Pot1pN was determined to consist of six nucleotides, GGTTAC, by gel filtration chromatography and filter-binding assay (K(D) = 83 nM). Pot1pN is a monomer, and each monomer binds one hexanucleotide. Experiments with nucleotide substitutions demonstrated that the central four nucleotides are crucial for binding. The dependence of Pot1pN-ssDNA binding on salt concentration was consistent with a single ionic contact between the protein and the ssDNA phosphate backbone, such that at physiological salt condition 83% of the free energy of binding is nonelectrostatic. Subsequent binding experiments with longer ssDNAs indicated that Pot1pN binds to telomeric ssDNA with 3' end preference and in a highly cooperative manner that mainly results from DNA-induced protein-protein interactions. Together, the binding properties of Pot1pN suggest that the protein anchors itself at the very 3' end of a chromosome and then fills in very efficiently, coating the entire single-stranded overhang of the telomere.

Animals↗