PubMed Health⌕ Search

Biomedical subjects

Da Xing

Publications and source records attributed to Da Xing.

At least 19 recordsLinked to original sources

A new principle photosynthesis capacity biosensor based on quantitative measurement of delayed fluorescence in vivo.

Delayed fluorescence (DF) is an excellent marker for evaluating plant photosynthesis. Compared with common methods for measuring the photosynthesis rate based on consumption of CO(2), DF technique can quantify the plant photosynthesis capacity more accurately and faster under its physiological status with less interference from the environment. We previously reported a method for measuring photosynthesis using DF of chloroplast [Wang, C.L., Xing, D., Chen, Q., 2004. Biosens. Bioelectron. 20, 454-459]. In the study, a novel fast and portable photosynthesis capacity biosensor system was developed, which was composed of light-emitting diode lattice as excitation light source, Channel Photomultiplier DC-Module to achieve DF, single-chip microcomputer as control center, hermetic dark sample chamber, battery power supply and CO(2), humidity and temperature controller. Compared with our previous work, the system was portable and can directly measure plant photosynthesis capacity in vivo in less than 10s. A database in the software to carry out data acquisition and processing was developed to translate maximal DF intensity to net photosynthesis rate (Pn). In addition, local-control and remote-control mode can be chosen in the system. To demonstrate the utility of the system, it was applied to evaluate maximum Pn of four different plant species samples (Queen Rape Myrtle (var. rubra), soybean (Lu Hei No. 1), maize (Jin Dan No. 39) and rice (Jing Dao No. 21)) in field. The results were compared with that using commercial photosynthesis system LI-6400 and the uncertainty was less than +/-5%. The new principle of photosynthesis measurement is a challenge and breakthrough to conventional method of gas exchange and may be a potential technique of next generation photosynthesis measurement.

Biosensing Techniques↗

Fluorescence resonance energy transfer analysis of bid activation in living cells during ultraviolet-induced apoptosis.

Ultraviolet (UV) irradiation is a DNA-damaging agent that triggers apoptosis through both the membrane death receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic Bcl-2 family member, is important in most cell types to apoptosis in response to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, comprised of yellow and cyan fluorescent protein and a full length Bid, was used as a fluorescence resonance energy transfer analysis (FRET) probe. Using the FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated at 9+/-1 h after UV irradiation, and the average duration of the activation was 75+/-10 min. Bid activation coincided with a collapse of the mitochondrial membrane potential with an average duration of 50+/-10 min. When cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the process of Bid activation was completely inhibited, but the apoptosis was only partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp specific inhibitor) did not block Bid activation. Furthermore, the endogenous Bid activation with or without Z-IETD-fmk in response to UV irradiation was confirmed by Western blotting. In summary, using the FRET technique, we observed the dynamics of Bid activation during UV-induced apoptosis and found that it was a caspase-8 dependent event.

Apoptosis↗

Effects of IkappaBalpha and its mutants on NF-kappaB and p53 signaling pathways.

AIM: To study the effects of IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) on NF-kappaB, p53 and their downstream target genes. The relationship of NF-kappaB, p53, and IkappaBalpha was further discussed. METHODS: pECFP-IkappaBalpha, pECFP-IkappaBalphaM (amino acides 1-317, Ser32, 36A), pECFP-IkappaBalpha243N (amino acides 1-243), pECFP-IkappaBalpha244C (amino acides 244-317), pEYFP-p65 and pp53-DsRed were constructed and transfected to ASTC-alpha-1 cells. Cells were transfected with pECFP-C1 as a control. 30 h after the transfection, location patterns of NF-kappaB, p53 and IkappaBalpha (IkappaBalphaM, IkappaBalpha243N, IkappaBalpha244C) were observed by a laser scanning microscope (LSM510/ConfoCor2, Zeiss). RNA extraction and reverse transcription were performed in cells transfected or co-transfected with different plasmids. Effects of IkappaBalpha and its mutants on the transprition level of NF-kappaB, NF-kappaB downstream target gene TNF-alpha, p53 and p53 downstream target gene Bax were observed by real time QT-PCR. In all experiments beta-actin was reference. Results are expressed as the target/reference ratio of the sample divided by the target/reference ratio of the control. Different transfected cells were incubated with CCK-8 for 2 h in the incubator. Then the absorbance at 450 nm was measured by using a microplate reader. RESULTS: Cells that were transfected with p53-DsRed revealed a predominant nuclear localization. YFP-p65 mainly existed in the cytoplasm. Cells were transfected with CFP-IkappaBalpha, CFP-IkappaBalphaM, and CFP-IkappaBalpha243N respectively and revealed a predominant cytosolic localization. However, cells transfected of CFP-IkappaBalpha244C revealed a predominant nuclear localization. The mRNA levels of p65, TNF-alpha, p53 and Bax in CFP-IkappaBalpha transfected cells did not change significantly, while in YFP-p65/CFP-IkappaBalpha co-transfected cells, IkappaBalpha decreased the transcription of p65 downstream gene TNF-alpha (2.24+/-0.503) compared with the YFP-p65/CFP-C1 co-transfected cells (5.08+/-0.891) (P<0.05). Phosphorylation defective IkappaBalpha (IkappaBalphaM) decreased the transcription levels of all the four genes compared with the control (P<0.05). The N terminus of IkappaBalpha (IkappaBalpha243N) increased the transcription of NF-kappaB (1.84+/-0.176) and TNF-alpha (1.51+/-0.203) a little bit. However, the C terminus of IkappaBalpha (IkappaBalpha244C) increased the transcription of NF-kappaB, TNF-alpha, p53 and Bax significantly (8.29+/-1.662, 14.16+/-2.121, 10.2+/-0.621, 3.72+/-0.346) (P<0.05). The CCK-8 experiment also showed that IkappaBalpha244C and p53 synergistically mediate apoptosis. CONCLUSIONS: IkappaBalpha and its mutants (IkappaBalphaM, IkappaBalpha243N, IkappaBalphaM244C) have different effects on NF-kappaB and p53 signaling pathways, according to their different structures. IkappaBalphaM bounds with NF-kappaB and p53 in cytoplasm steadily, and inhibits both of the two signaling pathways. p53 and IkappaBalpha244C may be co-factor in inducing apoptosis. The C terminal of IkappaBalpha enhanced cell death, which suggests that it may be a pro-apoptotic protein existed in cells.

Apoptosis↗

Single cell FRET imaging for determination of pathway of tumor cell apoptosis induced by photofrin-PDT.

Apoptosis is an important cellular event that plays a key role in pathogeny and therapy of many diseases. Apoptosis has been associated with photodynamic therapy (PDT) and its pathway is important in the mechanistic study of PDT. We show that single cell fluorescent imaging can be used to determine the pathway of PDT-induced tumor cell apoptosis. In this study, ASTC-a-1 tumor cells transfected by plasmid DNA SCAT3 were treated by Photofrin-PDT. The intracellular distribution of Photofrin was observed using a confocal microscope. The activations of caspase-3 and caspase-8 were dynamically observed using fluorescence resonance energy transfer (FRET). Our experimental results show that the Photofrin molecules are localized in cell mitochondria, and that after PDT caspase-3 was activated rapidly while caspase-8 remained inactive. These results demonstrate that the tumor cell apoptosis induced by Photofrin-PDT was directly initiated from the mitochondrial pathway.

Apoptosis↗

Single cell analysis of PKC activation during proliferation and apoptosis induced by laser irradiation.

Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.

Apoptosis↗

[Spectroscopic research on the mechanism for 730 nm component in delayed fluorescence of chloroplast].

Charge recombination in reaction center of photosystem II (PS II) is regarded as the origination of delayed fluorescence (DF). The mechanism for 730 nm component appearing in the DF spectrum of chloroplast was studied with different spectral analysis methods. Experimental results of the delayed fluorescence spectrum at different chloroplast concentration showed that the apexes at 685 and 730 nm ascend when the concentration is relative low; the peak value at 685 nm reaches a maximum when the concentration is 7.8 microg x m(L-1), however, the apex at 730 nm is still increasing. The peak value at 730 nm finally reaches a maximum at chloroplast concentration of 31.2 microg x mL(-1), while the apex at 685 nm has apparently decreased. The results of absorption spectrum showed that the ratios of A685 to A730 are almost a constant during the process of increasing chloroplast concentration. Furthermore, the excitation spectrum for 730 nm fluorescence shows that the 685 nm light has a high excitation efficiency. Above experimental results indicated that the 730 nm component of DF spectrum is the fluorescence of chlorophyll a in PSI reaction center excited by 685 nm DF. Meanwhile, this conclusion was further verified by the invariability of DF spectrum at different delay time (1-9 s).

Chlorophyll↗

[Quantitative determination of imidacloprid by infrared absorption spectrometry].

The content of imidacloprid in pesticide is usually measured by high-performance liquid chromatography, which is precise but time-consuming. In the present work the content of imidacloprid in pesticide was measured by infrared spectroscopy with pressing potassium bromide troche (KBr). A comparison of infrared spectra between analytic imidacloprid and commercial pesticide showed that the absorption peak of imidacloprid at 939.2 cm(-1) is independent and can be used as the characteristic absorption peak for quantitative determination. The area from 947 to 925.8 cm(-1) for the infrared spectra of imidacloprid and its content meet the linear equation: Area = l.3665 x 10(-1) + 2.37 x 10(-2) x c, r = 0.99953. Finally, the authors could draw a conclusion that the method of NIR, instead of routine lab analysis, is feasible for the determination of imidacloprid in pesticide.

Imidazoles↗

Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis.

In many apoptosis pathways, activation of caspase-3 is considered the final stage. In this study, we studied PDT induced caspase-3 activation with fluorescence resonance energy transfer (FRET) technique. A recombinant caspase-3 substrate, SCAT3, was used as the FRET probe. FRET fluorescence images were collected after PDT or TNF-alpha induced apoptosis. By analyzing the dynamic changes of FRET fluorescence, the results indicate that the caspase-3 activation started immediately after the PDT treatment. In contrast, FRET disruption caused by caspase-3 activation started at 3h after TNF-alpha treatment, due to different signaling pathway. The results have proofed, for the first time, that FRET is a sensitive technique that can be used to investigate PDT-induced activation of caspase-3 in real-time and in single cells. By choosing appropriate recombinant substrates as FRET probes, it is likely that FRET technique will provide a new real-time means to study the mechanism of PDT at single cell level.

Adenocarcinoma↗

Determination of optical properties of normal and adenomatous human colon tissues in vitro using integrating sphere techniques.

AIM: The purpose of the present study is to compare the optical properties of normal human colon mucosa/submucosa and muscle layer/chorion, and adenomatous human colon mucosa/submucosa and muscle layer/chorion in vitro at 476.5, 488, 496.5, 514.5 and 532 nm. We believe these differences in optical properties should help differential diagnosis of human colon tissues by using optical methods. METHODS: In vitro optical properties were investigated for four kinds of tissues: normal human colon mucosa/submucosa and muscle layer/chorion, and adenomatous human colon mucosa/submucosa and muscle layer/chorion. Tissue samples were taken from 13 human colons (13 adenomatous, 13 normal). From the normal human colons a total of 26 tissue samples, with a mean thickness of 0.40 mm, were used (13 from mucosa/submucosa and 13 from muscle layer/chorion), and from the adenomatous human bladders a total of 26 tissue samples, with a mean thickness of 0.40 mm, were used (13 from mucosa/submucosa and 13 from muscle layer/chorion). The measurements were performed using a double-integrating-sphere setup and the optical properties were assessed from these measurements using the adding-doubling method that was considered reliable. RESULTS: The results of measurement showed that there were significant differences in the absorption coefficients and scattering coefficients between normal and adenomatous human colon mucosa/submucosa at the same wavelength, and there were also significant differences in the two optical parameters between both colon muscle layer/chorion at the same wavelength. And there were large differences in the anisotropy factors between both colon mucosa/submucosa at the same wavelength, there were also large differences in the anisotropy factors between both colon muscle layer/chorion at the same wavelength. There were large differences in the value ranges of the absorption coefficients, scattering coefficients and anisotropy factors between both colon mucosa/submucosa, and there were also large differences in these value ranges between both colon muscle layer/chorion. There are the same orders of magnitude in the absorption coefficients for four kinds of colon tissues. The scattering coefficients of these tissues exceed the absorption coefficients by at least two orders of magnitude. CONCLUSION: There were large differences in the three optical parameters between normal and adenomatous human colon mucosa/submucosa at the same laser wavelength, and there were also large differences in these parameters between both colon muscle layer/chorion at the same laser wavelength. Large differences in optical parameters indicate that there were large differences in compositions and structures between both colon mucosa/submucosa, and between both colon muscle layer/chorion. Optical parameters for four kinds of colon tissues are wavelength dependent, and these differences would be useful and helpful in clinical applications of laser and tumors photodynamic therapy (PDT).

Adenomatous Polyposis Coli↗

Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation.

BACKGROUND AND OBJECTIVES: Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing wound etc. Yet, the mechanism of LPLI remains unclear. In order to determine the effects of high fluence LPLI on cell growth and caspase-3 activity, we have measured the dynamics of caspase-3 activity during cell apoptosis induced by high fluence LPLI treatment. STUDY DESIGN/MATERIALS AND METHODS: He-Ne laser was used to irradiate human lung adenocarcinoma cells (ASTC-a-1). Cell Counting Kit-8 was used for cytotoxicity assay. A fluorescent microscope was used to perform fluorescence resonance energy transfer (FRET) imaging. A luminescence spectrometer was used to acquire the fluorescent emission spectrum. Statistical analysis was performed with Student's paired t-test. RESULTS: Cytotoxicity assay showed that when light irradiation fluence exceeded 60 J/cm2, LPLI treatment induced ASTC-a-1 cell apoptosis in a fluence-dependent manner. FRET imaging and spectrofluorometric analysis demonstrated that caspase-3 was activated during high fluence LPLI-induced cell apoptosis. CONCLUSIONS: Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.

Adenocarcinoma↗

[Spectral properties of hematoporphyrin derivative after interacted with human serum albumin].

The spectral properties of photosensitizer HpD, human serum albumin (HSA) and their complex have been studied. The results show that HpD can form HpD-HSA complex with HSA in physiological condition. Compared with pure HpD, the maximum absorption and the fluorescence peaks for HpD-HSA complex had 8-10 nm red-shift. When HpD-HSA complex was excited by the light of corresponding to excitation peaks of HSA (228 and 279 nm) and HpD (394 nm), it was found that the absorption of HSA and HpD both contributed to the emission of HpD-HSA complex at 622 nm. The complex of HpD-HSA was individually excited by wavelengths corresponding to HpD absorption peaks of 402, 502, 537 and 570 nm, and the excitation efficiency of HpD-HSA at pumping wavelength of 537 and 570 nm was higher than that of HpD. The results demonstrate that the red-shift caused by the interaction of HpD and the special proteins in blood should be considered in selecting the excitation and emission wavelength in photodynamic diagnosis and therapy. The results also indicate that the excitation efficiency of porphyrin-protein complex in vivo is higher than that of HpD in vitro when a longer wavelength light corresponding to the week absorption peaks of porphyrin-protein complex is used in photodynamic therapy.

Hematoporphyrin Derivative↗

A method to quantitatively detect H-ras point mutation based on electrochemiluminescence.

Conventional methods for point mutation detection are usually multi-stage, laborious, and need to use radioactive isotopes or other hazardous materials, and the assay results are often semi-quantitative. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electrochemiluminescence (ECL) assay was coupled with restriction endonuclease digestion directly from PCR products. Only the wild-type amplicon containing the endonuclease's recognition site can be cut off, and thus cannot be detected by ECL assay. Using the PCR-ECL method, 30 bladder cancer samples were analyzed for possible point mutation at codon 12 of H-ras oncogene. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than three orders of magnitude. The point mutation was found in 14 (46.7%) out of 30 bladder cancer samples. The experiment results demonstrate that the PCR-ECL method is a feasible quantitative approach for point mutation detection due to its safety, high sensitivity, and simplicity.

Calibration↗

High sensitive approach for point mutation detection based on electrochemiluminescence.

An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for point mutation detection has been developed. The target is amplified using a tris (bipyridine) ruthenium (TBR)-labeled forward and a biotinylated reverse primer. The amplification products are digested with specific restriction enzyme, then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. The established technique was further applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the system has a low detection limit of 100 fmol and a linear range of more than 3 orders of magnitude for H-ras amplicon; the two genotypes can be reliably discriminated. In summary, the mutant specific ECL-PCR method can be used to detect a point mutation that creates or destroys a restriction site in any gene. It is useful in single nucleotide polymorphism (SNP) and mutation detection due to its safety, high sensitivity and simplicity.

Biomarkers, Tumor↗

Detection of genetically modified organisms by electrochemiluminescence PCR method.

With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (CaMV35S) promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.

Carica↗

A novel method for measuring photosynthesis using delayed fluorescence of chloroplast.

Photosynthesis is the most important chemical reaction in the world. The measurement of plant photosynthesis rate plays an important role in agriculture. Light-induced delayed fluorescence (DF) in plants is an intrinsic label of the efficiency of charge separation at P680 in photosystem II (PS II). In this paper, we have developed a biosensor that can accurately measure the plant photosynthesis ability by means of DF. Compared with common methods for measuring the photosynthesis rate based on consumption of CO2, the proposed technique can quantify the plant photosynthesis ability with less influence of the environment. The biosensor is an all-weather measuring instrument, it has its own illumination power and utilizes intrinsic DF as the measurement marker. The current investigation has revealed that, there is a good correspondence between the results measured by the biosensor and that by commercially available portable photosynthesis system under controlled conditions. We thus conclude that DF is an excellent marker for evaluating plant photosynthesis ability under its biological status with less interferences of the environment.

Biosensing Techniques↗

Photoacoustic imaging with deconvolution algorithm.

The impulse response of the ultrasonic transducer used for detection is crucial for photoacoustic imaging with high resolution. We demonstrate a reconstruction method that allows the optical absorption distribution of a sample to be reconstructed without knowing the impulse response of the ultrasonic transducer. A convolution relationship between photoacoustic signals measured by an ultrasound transducer and optical absorption distribution is developed. Based on this theory, the projection of the optical absorption distribution of a sample can be obtained directly by deconvolving the recorded PA signal originating from a point source out of that from the sample. And a modified filtered back projection algorithm is used to reconstruct the optical absorption distribution. We constructed a photoacoustic imaging system to validate the reconstruction method and the experimental results demonstrated that the reconstructed images agreed well with the original phantom samples. The spatial resolution of the system reaches 0.3 mm.

Acoustics↗

Fast photoacoustic imaging system based on 320-element linear transducer array.

A fast photoacoustic (PA) imaging system, based on a 320-transducer linear array, was developed and tested on a tissue phantom. To reconstruct a test tomographic image, 64 time-domain PA signals were acquired from a tissue phantom with embedded light-absorption targets. A signal acquisition was accomplished by utilizing 11 phase-controlled sub-arrays, each consisting of four transducers. The results show that the system can rapidly map the optical absorption of a tissue phantom and effectively detect the embedded light-absorbing target. By utilizing the multi-element linear transducer array and phase-controlled imaging algorithm, we thus can acquire PA tomography more efficiently, compared to other existing technology and algorithms. The methodology and equipment thus provide a rapid and reliable approach to PA imaging that may have potential applications in noninvasive imaging and clinic diagnosis.

Acoustics↗

Rapid and sensitive immunomagnetic-electrochemiluminescent detection of p53 antibodies in human serum.

The mutation of tumor suppressor p53 gene is common in malignant tumor. p53 antibodies are products of immunoresponse against abnormal p53 protein. It has been found that p53 antibodies are of importance in tumor's diagnosis, prognosis and relapse monitoring. However, current method for detecting p53 antibodies, i.e. enzyme-linked immunosorbent assay (ELISA), requires a long time with multiple steps, and the assay is only semi-quantitative. In this work, a protocol for quantitative detection of p53 antibodies in human serum using immunomagnetic electrochemiluminescence (IM-ECL) was devoloped. The immunoassay format consisted of a three antibody sandwich in which a biotinylated capture antibody, was banded with the commercial p53 protein. A detector antibody was added to bind the p53 protein at another site. Then, secondary antibody, labeled with ruthenium(II) tris-bipyridal, was added and, when bound to the bead immunocompiex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the analyzer made by us. Our experimental results indicate that the sensitivity of this assay was 10 pg of p53 antibodies per ml of reference serum (normal human serum). A stable calibration curve with a wide dynamic range was established. The calibration curve was linear from 0.01 to 1000 ng/ml, thus, making quantitation possible. An immunologic prozone effect was observed above 1000 ng p53 antibodies per milliliter of serum. Serum samples from lung and nasopharyngeal carcinoma patients were tested using the IM-ECL assay. The positive rate of p53 antibodies were 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. p53 antibody concentration in the carcerous human sera were quantified from the calibration curve. In the case of lung carcinoma, a trend was found that a higher p53 antibody concentration in the serum was likely linked to a higher stage of the cancer. This trend was not found in nasopharyngeal carcinoma. The assay uses only 50 microl of sample per test and requires a 30-min incubation period in addition to a 50 s acquisition time. This assay has several advantages over the commonly used ELISA method in terms of sensitivity, linear range, and assay time. Results of the study suggest that IM-ECL is a feasible method for rapid and sensitive detection of p53 antibodies in human serum.

Antibodies↗