Biomedical subjects

Fang Yu

Publications and source records attributed to Fang Yu.

35 records · Page 2Linked to original sources

PubMed2004

Surface plasmon fluorescence immunoassay of free prostate-specific antigen in human plasma at the femtomolar level.

This study describes the development of a surface plasmon fluorescence spectroscopy (SPFS)-based sandwich immunoassay, for the detection of free prostate-specific antigen (f-PSA). The commercial CM5 chip (Biacore) was integrated into the SPFS-based assay making use of its three-dimensionally extended dextran architecture, which offers a large protein retention capacity and alleviates the metal-induced fluorescence quenching. The performance of the f-PSA assay was investigated in buffer and in human plasma, respectively. In the human plasma, the limit of detection of f-PSA was estimated to be approximately 80 fM for 40 min of contact time, which adequately meets clinical requirements.

Humans↗

PubMed2004

Attomolar sensitivity in bioassays based on surface plasmon fluorescence spectroscopy.

With the aid of a dextran matrix, the metal-induced fluorescence loss of bound fluorophores can be greatly reduced, and the distance dependence of the fluorescence yield could be largely convoluted. This is optimized for the limit of detection assessment of surface plasmon fluorescence spectroscopy. The model system was designed as a direct assay with mouse IgG covalently immobilized to the carboxymethyl dextran matrix of a CM5 sensor chip from Biacore. Time-resolved ultratrace detection of fluorophore (Alexa-Fluor 647)-labeled rabbit anti-mouse antibody down to 500 aM (10-18 M) was accomplished, corresponding to a binding rate of approximately 10 molecules mm-2 min-1.

Animals↗

PubMed2004

Surface plasmon enhanced diffraction for label-free biosensing.

Surface plasmon enhanced evanescent field at a (noble) metal/dielectric interface can be employed to enhance the diffraction efficiency of surface grating structure composed of biomolecules. Based on a Kretschmann configuration, we realized a diffraction biosensor to monitor the dynamic interaction of biological molecules in a label-free way. It was demonstrated by the binding of an anti-biotin antibody to the biotin-functionalized region of a periodically patterned surface, which generated significant optical contrast to diffract the surface plasmon field. With the aid of the synchronic surface plasmon resonance signal, a quadratic dependence of diffraction signal on the amount of bound antibody was found, which coincides with the theoretical expectation. Time-dependent measurements were conducted to estimate the density of biotin thiols on the functional region.

Antibodies, Monoclonal↗

PubMed2004

Oligonucleotide hybridization studied by a surface plasmon diffraction sensor (SPDS).

A novel label-free biosensor concept based on surface plasmon-enhanced diffraction by micro- patterned interfaces was applied to the study of hybridization reactions of target DNA oligonucleotides (15mers and 75mers) from solution to probe DNA oligonucleotides attached via streptavidin to the sensor surface. The self-referencing and quadratic signal amplification mechanism of the sensor allowed highly sensitive detection of the hybridization process. Association and dissociation processes of DNA targets could be recorded in real time and used for the quantification of their binding affinities, which differ considerably with a single base pair mismatch. An equilibrium titration approach was also applied in order to obtain the binding affinities for 15mer targets, yielding similar affinity values. The hybridization efficiencies were found to be higher for the 15mers than for the 75mers, although the latter contained the same recognition sequences. The hybridization efficiency was shown to depend on the probe density and reached nearly 100% for the 15mer fully complementary targets at a probe density of approximately 1.2 x 10(12) molecules/cm2. Using the assay as an end-point determination method, the lowest detectable coverage of a 15mer oligonucleotide was at least approximately 1.1 x 10(11) molecules/cm2. The diffraction sensing concept offers a completely novel way to integrate a reference channel in large-scale, label-free screening applications, to improve the stability and to enhance the sensitivity of microarray read-out systems.

Base Pair Mismatch↗

PubMed2004

Evanescent field in surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopies.

The highly sensitive nature of surface plasmon resonance (SPR) spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS) are governed by the strong surface plasmon resonance-generated evanescent field at the metal/dielectric interface. The greatest evanescent field amplitude at the interface and the maximum attenuation of the reflectance are observed when a nonabsorbing dielectric is employed. An absorbing dielectric decreases the evanescent field enhancement at the interface. The SPR curve of an absorbing dielectric is characterized by a greater reflectance minimum and a broader curve, as compared to those of the nonabsorbing dielectric with the same refractive index. For a weakly absorbing dielectric, such as nanometer-thick surface-confined fluorophores, the absorption is too small to induce a significant change in the SPR curve. However, the presence of a minute amount of the fluorophore can be detected by the highly sensitive SPFS. The angle with the maximum fluorescence intensity of an SPFS curve is always smaller than the resonance angle of the corresponding SPR curve. This discrepancy is due to the differences of evanescent field distributions and their decay characteristics within the metal film and the dielectric medium. The fluorescence intensity in an SPFS curve can be expressed in terms of the evanescent field amplitude. Excellent correlations between the experimentally measured fluorescence intensities and the evanescent field amplitudes are observed.

Journal Article↗

PubMed2004

Immunosensor with self-referencing based on surface plasmon diffraction.

We propose a novel type of immunosensor based on the diffraction of surface plasmon, which allows for in situ, real-time, and label-free observation of interfacial binding events. The inherent self-referencing mechanism of surface diffraction was found to be very effective for compensating fluctuations of the bulk, demonstrated by a temperature variation experiment. Possessing a stable baseline signal, the diffraction sensor offered picomolar sensitivity in directly detecting the binding of human chorionic gonadotropin (hCG) hormone.

Journal Article↗

PubMed2004

[The relationship of angiotensin I-converting enzyme gene polymorphism with diabetic retinopathy and diabetes myocardial infarction].

OBJECTIVE: To investigate the relationship of angiotensin I-converting enzyme (ACE) gene polymorphism to diabetic retinopathy and diabetes myocardial infarction. METHODS: ACE insertion/deletion(I/D) polymorphism was determined by PCR. RESULTS: No evidence showed that ACE gene was associated with diabetic retinopathy. By comparison of the type 2 diabetes patients with myocardial infarction versus those without-myocardial infarction, it was found that the frequencies of homozygote DD (41.2% versus 33.2%) and of allele D (64.7% versus 55.0%) increased remarkably; the difference was statistically significant (P<0.05). CONCLUSION: Allele D(RR=1.50) and genotype DD(RR=1.33) seemed to be a genetic risk factor for type 2 diabetes myocardial infarction.

Adult↗

PubMed2004

[Treatment of high-risk neuroblastoma with intensive chemotherapy, autologous peripheral blood stem cell transplantation, and 13-cis-retinoic acid].

OBJECTIVE: The prognosis for neuroblastoma in advanced stage is still poor, even under conventional chemotherapy. This study aimed to investigate if very high dose chemotherapy in conjunction with autologous peripheral blood stem cell transplantation and 13-cis-retinoic acid could get excellent results in children with high risk neuroblastoma. METHODS: Six children, aged from 4 to 8 years, with stage IV neuroblastoma were included in the study. The duration of the illness before admission was 1 to 12 months. Primary sites of the diseases were in the abdominal cavity (n = 5) and thoracic cavity (n = 1). All of patients had bone marrow metastasis, and one had multiple bone metastasis and orbital metastasis. All of the patients received very high dose chemotherapy, surgery, local radiation (20-30 Gy), and autologous peripheral blood stem cell transplantation as well as 13-cis retinoic acid. Induction chemotherapy included vincristine 0.67 mg/(m2 x 24 h, x 3), cyclophosphamide 2.1 g/(m2 x 24 h, x 2) and doxorubicin 25 mg/(m2 x 24 h, x 3) for 4 courses. Drugs were given as 24 hour-continuous intravenous infusion. Etopside 200 mg/(m2 x 24 h, x 3) and cisplatin 50 mg/(m2 x 24 h, x 3) were given for 2 courses. Conditioning regimen included carboplatin 400 mg/(m2.d) for 4 days, etoposide 300 mg/(m2.d) for 4 days and melphalan 70 mg/(m2.d) for 3 days. 13-cis retinoic acid 160 mg/(m2.d) started on +59 days for 6 courses, each course including 14 days therapy and 14 days rest. RESULTS: Six patients got a complete response before stem cell transplantation. Their bone marrow metastasis disappeared and so did bone and orbital metastasis. However, marrow suppression due to very high dose chemotherapy occurred in all of the patients, which lasted for 3-4 weeks for peripheral leukocyte recovery. Fever occurred after they finished 1/3 course of chemotherapy. Infection, however, was cured with the use of Fortum and Imipenem, ect. Autologous peripheral blood stem cell transplantation was initiated and successful in all cases. Follow-up studies revealed that all the patients were in CR status 4-18 months after transplant, and the cardiac and liver and renal functions were normal. Meanwhile, bone marrow was recovered or in the process of recovery. CONCLUSION: The new strategies focused on very high dose chemotherapy, autologous peripheral blood stem cell transplantation and biological therapy might be a good option for patients with advance neuroblastoma.

Abdominal Neoplasms↗

PubMed2004

[Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma].

OBJECTIVE: To assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC). METHODS: Samples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR. RESULTS: CpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines. CONCLUSIONS: Our results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.

Adaptor Proteins, Signal Transducing↗

PubMed2004

[Genetic polymorphisms of STR loci THO1, TPOX, CSF1PO in Guizhou Han population].

To understand the genetic polymorphism at THO1, TPOX, CSF1PO STR loci for Han population in Guizhou Province, and construct a preliminary database, EDTA-blood specimens were collected from the 110 unrelated individuals in Han population from Guizhou. The DNA samples were extracted with Chelex method and amplified by multiplex polymerase chain reaction. The PAGE was used to type the PCR products. The allele frequencies were compared with other Han populations. The genotype distributions of THO1, TPOX and CSF1PO were in accordance with Hardy-Weinberg equilibrium. The combined PD and PE were 0.9986 and 0.832 respectively. All of the three loci in this study provide useful marker for forensic paternity test and individual identification.

Alleles↗

PubMed2003

Second-chance rearrangement route to novel 5(6)-syn,anti-difunctional 2-azabicyclo[2.1.1]hexanes.

[reaction: see text] The first syntheses of 5,6-difunctionalized-2-azabicyclo[2.1.1]hexanes containing syn-hydroxy and syn-fluoro substituents have been effected in a stereocontrolled manner. The key reactions are regioselective additions to the aziridinium ions formed from 6-exo-iodo(bromo)-5-endo-X-2-azabicyclo[2.2.0]hexanes (X = F, OH) upon silver or mercury salt enhancement of iodide nucleofugacity.

Journal Article↗

PubMed2003

Surface plasmon field-enhanced fluorescence spectroscopy studies of the interaction between an antibody and its surface-coupled antigen.

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface plasmon mode for the excitation of surface-confined fluorophores. The ability to simultaneously monitor the interfacial refractive index changes and the fluorescence signals in real time offers a huge potential for applications of SPFS in surface immunoreaction detection. In this study, gold surfaces were functionalized by mixed self-assembled monolayers exposing an antigen (biotin) at a density that was varied over a wide range. Specific antibody-antigen interactions were observed for anti-biotin antibody solutions passing over the surfaces with a rather high flow speed driven by a home-built liquid-handling system. First, the use of the fluorophores Cy5 and Alexa Fluor 647 in SFPS-based immunoassays was investigated. It was found that Cy5 exhibits strong self-quenching, which makes it rather unsuitable for quantitative measurements. For the in situ measurement of the binding kinetics, an angular "detuning" effect was confirmed to negatively interfere with the fluorescence signal in cases where large SPR signals were detected. An in-depth comparison between the SPR and the fluorescence signal reveals that the fluorescence yield of the dyes depends strongly on the separation distance from the gold surface. And finally, we stress the ability of SPFS to detect binding to surfaces containing extremely diluted antigen density, where the SPR signal failed to follow.

Animals↗

PubMed2003

The synthesis of SO-3, a conopeptide with high analgesic activity derived from Conus striatus.

The synthesis and characterization of the conopeptide, SO-3, originally derived from Conus striatus is reported. It contains 25 amino acid residues and three disulfide bridges and manifests 72% sequence identity with MVIIA, an N-type Ca2+ channel inhibitor of high analgesic activity. We evaluated SO-3 in several mouse models of pain. The results indicate that SO-3 is a potent, nonaddictive, analgesic agent.

Amino Acid Sequence↗

PubMed2003

[Genetic polymorphisms of short tandem repeats loci F13A01, FESFPS, vWA from Han population in Guizhou].

OBJECTIVE: To understand the allele structure and genetic polymorphism at F13A01, FESFPS and vWA short tandem repeats (STRs) loci for Han population in Guizhou province of China, and to construct a preliminary database. METHODS: One hundred sixty unrelated individuals in Han population from Guizhou were analyzed by multiplex polymerase chain reaction. The resultant allele frequencies were compared with those of other population or race database. RESULTS: The genotype distributions of F13A01, FESFPS and vWA were in accordance with Hardy-Weinberg equilibrium. The combined discrimination power and polymorphism information content were 0.9984 and 0.9700 respectively. CONCLUSION: All of the three loci in this study provide useful markers for forensic paternity test and for genetic purposes.

Alleles↗

PubMed2003

[Screening for lifespan-extension mutants with paraquat in Arabidopsis].

Genetic analyses of lifespan in model animals have revealed that extended lifespans are closely associated to increased resistance to oxidative stress. In the model plant Arabidopsis, late-flowering mutants are also found to be more tolerant to oxidative stress. However, Arabidopsis mutants with extended lifespans are poorly studied so far. In this study, a screening system for mutants with extended lifespans in Arabidopsis was established using paraquat, a potent herbicide that exacerbates O2-. radical production. The relationship between lifespan and resistance to oxidative stress was examined with one of the mutants, SFNA-9-4. Compared to that of wild type, the lifespan of SFNA-9-4 is significantly extended, and its resistance to oxidative stress is also significantly elevated. These results suggest that, as in Drosophila, paraquat can also be used to screen for mutants with extended lifespans in Arabidopsis.

Animals↗

PubMed2002

Impaired renal Na(+) retention in the sgk1-knockout mouse.

The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na(+) channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1(-/-)) mice and wild-type (sgk1(+/+)) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1(-/-) mice to adequately decrease Na(+) excretion despite increases in plasma aldosterone levels and proximal-tubular Na(+) and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate.

Animals↗

PubMed2002

[Construction of eukaryotic expression vector using neomycin-resistance gene mutant as selectable marker].

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.

Amino Acid Sequence↗