Biomedical subjects

Quan Chen

Publications and source records attributed to Quan Chen.

At least 55 records · Page 3Linked to original sources

PubMed2004

[Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein].

OBJECTIVE: To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis. METHODS: The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid. The recombinant CFP10-ESAT-6 fusion protein was expressed and purified. Its antigenicity was confirmed by Western blot. Animal models infected with M. tuberculosis H(37)Rv strain and M. bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis. RESULTS: The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence. The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein. The purity and concentration of the final product was 98% and 1.2 g/L, respectively. Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively. The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA. Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG. CONCLUSIONS: The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E. coli. It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M. tuberculosis H(37)Rv strain from immunization with M. bovis BCG. The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis.

Animals↗

PubMed2004

Noise reduction using spatial-angular compounding for elastography.

Ultrasound elastography has developed into an imaging modality suitable for detection and diagnosis of cancers in the breast, prostate, and thyroid and for monitoring ablative therapies in the liver, kidneys, and other sites. In this article, a new approach is described that enables the reduction of noise artifacts in elastography without a significant reduction in either the contrast or spatial resolution. The technique uses angular-weighted compounding of local angular strains estimated from echo signals scanned at different insonification angles. Strain estimated along angular insonification directions can be separated into strain tensor components along the axial (direction of compression) and lateral directions. The mechanical stimulus is applied only along one direction. Angular-weighting factors are derived from the relationship between the axial and lateral strains under the assumption of tissue incompressibility. Experimental results using a uniformly elastic, tissue-mimicking phantom demonstrate the improvement in the signal-to-noise ratio obtained with angular-weighted compounding. Variation in the signal-to-noise ratio obtained using different angular increments also is investigated. Elastograms obtained from an inclusion phantom also demonstrate the improvement in contrast detail resolution obtained using spatial-angular compounding.

Algorithms↗

PubMed2004

[A study of the protective effect of the DNA vaccine encoding tubercle antigen 85B with MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To explore the protective effect of the DNA vaccine encoding Mycobacterium tuberculosis Ag85B with MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: Fifty four C57BL/6 mice were randomized into six groups and subjected to the following treatments respectively: intramuscularly immunized with PBS, pcDNA3.1, BCG, pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/Ag85B + pcDNA/MPT64 on three occasions at 3-week intervals. The BCG group received a single subcutaneous injection of 1 x 10(6) CFU BCG. The mice were challenged with 10(6) CFU H(37)Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed, and the number of CFU in organs and histopathologic changes were determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: The number of bacterial colonies in the lungs and spleens were lg(-1) (7.854 +/- 0.003) CFU/g and lg(-1) (7.190 +/- 0.016) CFU/g in PBS group, lg(-1) (7.700 +/- 0.016) CFU/g and lg(-1) (7.072 +/- 0.068) CFU/g in pcDNA3.1 group, lg(-1) (6.449 +/- 0.002) CFU/g and lg(-1) (5.436 +/- 0.042) CFU/g in BCG group, lg(-1) (7.370 +/- 0.002) CFU/g and lg(-1) (6.430 +/- 0.009) CFU/g in pcDNA/Ag85B group, lg(-1) (7.547 +/- 0.003) CFU/g and lg(-1) (6.784 +/- 0.002) CFU/g in pcDNA/MPT64 group, and lg(-1) (6.918 +/- 0.002) CFU/g and lg(-1) (6.079 +/- 0.004) CFU/g in pcDNA/Ag85B + pcDNA/MPT64 group respectively, which showed significant decrease at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/Ag85B group was higher than that of the other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B + pcDNA/MPT64 group was higher than that of the other groups (P < 0.05). No IL-4 was detected in all groups. The pulmonary histopathological changes were observed 6 weeks later following challenge with Mycobacterium tuberculosis H(37)Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B group was characterized by serofibrous inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in the lung. The survival time of BCG vaccinated mice after challenge with Mycobacterium tuberculosis H(37)Rv was longer than that of the other groups. The survival time of pcDNA/Ag85B + pcDNA/MPT64 group was longer than that of other DNA vaccine groups. CONCLUSION: The protective effect of BCG was more significant than the other groups, while the effect of pcDNA/Ag85B + pcDNA/MPT64 was better than other DNA vaccines.

Acyltransferases↗

PubMed2004

[Construction and expression of the eukaryotic coexpression plasmid containing Ag85B gene of Mycobacterium tuberculosis and IL-12 gene].

AIM: To construct an eukaryotic coexpression plasmid containing Mycobacterium tuberculosis (MTB) Ag85B and IL-12 genes. METHODS: MTB Ag85B gene and IL-12 gene were cloned into pBudCE4.1 which has multiple promoters to construct recombinant plasmid pBud85B-IL12. The recombinant plasmid was transfected into COS-7 cells and the expression of target genes was assessed by RT-PCR and ELISA. RESULTS: The expressions of Ag85B and IL-12 could be detected in COS-7 cells. CONCLUSION: The recombinant plasmid pBud85B-IL12 was constructed and expressed successfully, which lays the foundation for further development of DNA vaccine against tuberculosis.

Acyltransferases↗

PubMed2004

[The apoptosis-inducing activity of human selenoprotein P shorter isoform].

OBJECTIVE: Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro. METHODS: The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied. RESULTS: The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors. CONCLUSION: HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.

Animals↗

PubMed2003

Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells.

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.

Animals↗

PubMed2003

Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins.

Bcl-2 family of proteins plays differential roles in regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to cytosol. Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), ADP/ATP exchange, or oxidative phosphorylation during apoptosis. Although the mitochondrial homeostasis is affected by the relative ratio of pro- and anti-apoptotic Bcl-2 family members, the molecular mechanism underlying the release of mitochondrial intermembrane proteins remains elusive. Here we reported the biochemical evidence that both pro-apoptotic Bax and anti-apoptotic Bcl-X(L) might simultaneously contact the putative loop regions of human VDAC1, and the existence of VDAC1-Bax-Bcl-X(L) tertiary complex in vitro suggested that VDAC1 channel conformation and mitochondrial permeability could be determined by the delicate balance between Bax and Bcl-X(L).

Binding Sites↗

PubMed2003

One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1.

The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.

Chromatography, Affinity↗

PubMed2003

Role of Apo2L/TRAIL and Bcl-2-family proteins in apoptosis of multiple myeloma.

Apo2 Ligand or Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) is a member of the TNF gene superfamily that selectively induces apoptosis in tumor cells of diverse origins through engagement of death receptors. We have recently demonstrated that Type I interferons (IFN-alpha and beta) induce apoptosis in multiple myeloma (MM) cell lines and in plasma cells from MM patients. Moreover, Apo2L selectively induces apoptosis of patient MM tumor cells while sparing non-malignant cells. Apo2L induction is one of the earliest events following IFN administration in these cells. IFNs activate Caspases and the mitochondrial-dependent apoptotic pathway mediated by Apo2L production. Cell death induced by IFNs and Apo2L can be blocked by a dominant-negative Apo2L receptor, DRS, and is regulated by members of the Bcl-2 family of proteins. This review is focused on the apoptotic signaling pathways regulated by Apo2L and Bcl-2-family proteins and summarizes what is known about their clinical role.

Apoptosis↗

PubMed2003

Redox Regulation of Apoptosis before and after Cytochrome C Release.

Programmed cell death, or apoptosis, is one of the most studied areas of modern biology. Apoptosis is a genetically regulated process, which plays an essential role in the development and homeostasis of higher organisms. Mitochondria, known to play a central role in regulating cellular metabolism, was found to be critical for regulating apoptosis induced under both physiological and pathological conditions. Mitochondria are a major source of reactive oxygen species (ROS) but they can also serve as its target during the apoptosis process. Release of apoptogenic factors from mitochondria, the best known of which is cytochrome c, leads to assembly of a large apoptosis-inducing complex called the apoptosome. Cysteine proteases (called caspases) are recruited to this complex and, following their activation by proteolytic cleavage, activate other caspases, which in turn target for specific cleavage a large number of cellular proteins. The redox regulation of apoptosis during and after cytochrome c release is an area of intense investigation. This review summarizes what is known about the biological role of ROS and its targets in apoptosis with an emphasis on its intricate connections to mitochondria and the basic components of cell death.

Journal Article↗

PubMed2003

Ultrasound attenuation imaging using compound acquisition and processing.

A method that combines both spatial and frequency compounding is described for measuring attenuation in tissue. The technique applies a reference phantom to account for imaging system dependencies of echo signals. Emphasis is given to local attenuation estimates, to reduce the variance of the attenuation measurements over small regions of interest (ROI) and to enable coarse attenuation imaging. Experiments using a uniform phantom show that the standard deviation of local attenuation estimates within a ROI drops when greater degrees of compounding are applied. Attenuation images of a specially designed phantom containing inclusions with attenuation contrast illustrate the accuracy and precision of the technique.

Phantoms, Imaging↗

PubMed2003

Elastographic measurement of the area and volume of thermal lesions resulting from radiofrequency ablation: pathologic correlation.

OBJECTIVE: Elastography is a promising tool for visualizing the zone of necrosis in liver tissue resulting from radiofrequency tumor ablation. Because heat-ablated tissues are stiffer than normal untreated tissue, elastography may prove useful for following up patients who undergo radiofrequency ablative therapy. We sought to report the initial evaluations of the reliability of elastography for delineating thermal lesion boundaries in liver tissue by comparing lesion dimensions determined by elastography with the findings at whole-mount pathology. MATERIALS AND METHODS: Radiofrequency ablation was performed in vitro on liver tissue specimens encased in gelatin phantoms. The imaging plane for elastography was perpendicular to the axis of the radiofrequency electrode so that the ablated region was around the center of the plane. To obtain three-dimensional visualization of thermal lesions, we reconstructed the lesions from multiple elastograms by linearly translating the elastographic scanning plane. Pathology photographs were obtained in the same image plane used for elastography by slicing through the gelatin and tissue phantom using external markers. We used digitized gross pathology photographs obtained at a specified slice thickness to compute the areas and volumes of the lesions. These measurements were then compared to the measurements obtained from the elastograms. RESULTS: In a sample of 40 thermal lesions, we obtained a correlation between in vitro elastographic and pathologic measurements of r = 0.9371 (p < 0.00001) for area estimates and r = 0.979 (p < 0.00001) for volume estimates. CONCLUSION: We found excellent correlation between the measurements of the dimensions, areas, and volumes of thermal lesions that were based on elastographic images and the measurements that were based on digitized pathologic images. When compared with digitized pathologic measurements, elastographic measurements showed a tendency to slightly underestimate both the areas and volumes of lesions. Nevertheless, elastography is a reliable technique for delineating thermal lesions resulting from radiofrequency ablation.

Animals↗

PubMed2003

Relationship between telomerase activity and its subunit expression and inhibitory effect of antisense hTR on pancreatic carcinoma.

AIM: To directly investigate the relationship between telomerase activity and its subunit expression and the inhibitory effect of antisense hTR on pancreatic carcinogenesis. METHODS: We examined the telomerase activity and its subunit expression by cell culture, polymerase chain reaction (PCR), PCR-silver staining, PCR-ELISA, DNA sequencing, MTT and flow cytometry methods. RESULTS: PCR-silver staining and PCR-ELISA methods had the same specificity and sensitivity as the TRAP method. Telomerase activity was detected in the extract of the 10(th),20(th) and 30(th) passages of P3 cells,while it was absent in fibroblasts. Furthermore, after the 30th generation, the proliferation period of fibroblast cells was significantly prolonged. Telomerase activity and hTERTmRNA were detected in two pancreatic carcinoma cell lines, but were found to be negative in human fibroblast cells. Telomerase activity and hTERTmRNA were tested in pancreatic carcinoma specimens of 24 cases. The telomerase activity was positive in 21 of the 24 cases (87.5 %), and the hTERTmRNA in 20 cases (83.3 %). In adjacent normal tissues positive rates were both 12.5 %. There was a significant difference between the two groups. This indicated a significant correlation between the expression level of telomerase activity and histologic differentiation, metastasis and advanced clinical stage of pancreatic carcinoma. Our findings showed that the expressions of hTR and TP1mRNA were not correlated with the activity of telomerase but the expression of hTERTmRNA was. After treatment with PS-ODNs, telomerase activity in P(3) cells weakened and the inhibiting effect became stronger with an increase in PS-ODNs concentration. There was a significant difference between different PS-ODN groups (P<0.05). Inhibition of telomerase activity occurred most significant with PS-ODN1. The results of the FCM test of pancreatic cancer P(3) cells showed an increase in the apoptotic rate with increasing PS-ODN1 and PS-ODN2 concentrations. CONCLUSION: The expression of telomerase activity has a significant relationship to carcinogenesis. A strong correlation exists between telomerase activity and hTERTmRNA expression. The up-regulation of hTERTmRNA expression may play a critical role in human carcinogenesis. The expression of telomerase activity and its subunit level in pancreatic carcinoma significantly correlate with the clinical stage of pancreatic carcinoma and hence, may be helpful in its diagnosis and prognosis. The anti-hTR complementary to the template region of hTR is sufficient to inhibit P3 cell telomerase activity and cell proliferation in vitro, and can lead to a profound induction of programmed cell death.

Carcinoma↗

PubMed2003

[Construction of the prokaryotic expression vector of MTB lhp gene and its expression].

AIM: To construct prokaryotic expression vector carrying lhp gene and express it in E.coli. METHODS: The MTB lhp gene was amplified by PCR and then cloned into plasmid pQE30. After sequencing, the gene was cloned into plasmid pET32a(+) to construct recombinant prokaryotic expression vectors pQE30-CFP10 and pET32a(+)-CFP10. RESULTS: After transformation of the E.coli and induction with 1 mmol/L of IPTG, no additional protein was expressed in pQE30-CFP10 system, but recombinant target protein with M(r) 20 000 or so was expressed in pET32a(+)-CFP10 system, and the expressed protein was maximum when induced with IPTG for 4 h. The expressed protein existed in cytoplasm in soluble form and amounted to 38% of total protein of E.coli. Western blot analysis showed that the protein had good antigenicity. The purity of the protein purified through the Ni-NTA resin reached 93%. CONCLUSION: The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully and the rCFP10 protein was obtained, which laid the foundation for application of the rCFP10.

Blotting, Western↗

PubMed2003

[Immunogenicity of DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64].

AIM: To study the immunogenicity of a DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64. METHODS: C57BL/6 mice were intramuscularly injected respectively with PBS(A group), plasmid vector (B group), pcDNA/Ag85B(C group), pcDNA/MPT64(D group), and pcDNA/Ag85B MPT64 (E group) for three times with 2 week intervals. The mice were sacrificed 4 weeks after the final immunization. The total specific antibody levels were detected by ELISA and simultaneously spleen lymphocyte proliferation response to PPD and IFN-gamma level in culture supernatant of the PPD stimulated spleen lymphocytes was measured by MTT colorimetry and ELISA, respectively. RESULTS: The Ag85B, MPT64 and fusion gene Ag85B MPT64 DNA vaccines could induce anti-PPD antibody production as well as antigen specific lymphocyte proliferation and high levels of IFN-gamma in immunized mice. CONCLUSION: The Ag85B MPT64 fusion gene can induce specific cellular immunity and humoral immunity in immunized mice.

Acyltransferases↗

PubMed2002

A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome.

The high degree of similarity between the mouse and human genomes is demonstrated through analysis of the sequence of mouse chromosome 16 (Mmu 16), which was obtained as part of a whole-genome shotgun assembly of the mouse genome. The mouse genome is about 10% smaller than the human genome, owing to a lower repetitive DNA content. Comparison of the structure and protein-coding potential of Mmu 16 with that of the homologous segments of the human genome identifies regions of conserved synteny with human chromosomes (Hsa) 3, 8, 12, 16, 21, and 22. Gene content and order are highly conserved between Mmu 16 and the syntenic blocks of the human genome. Of the 731 predicted genes on Mmu 16, 509 align with orthologs on the corresponding portions of the human genome, 44 are likely paralogous to these genes, and 164 genes have homologs elsewhere in the human genome; there are 14 genes for which we could find no human counterpart.

Animals↗

PubMed2002

A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro.

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase-3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl-xL, cyclosporin A and z-VAD.fmk. Moreover, caspase-3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl-xL- and cyclosporin A-inhibitable manner. These results suggest that caspase-3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Pressure-dependent attenuation in ultrasound contrast agents.

Although microbubble contrast agents are believed to respond differently under different driving-pressure amplitudes, few studies have been performed to extensively study the pressure-dependence of their properties. In this paper, attenuation coefficients of two contrast agents (Optison and Definity) were measured under different driving-pressure amplitudes using a narrowband incident pulse. The attenuation of both contrast agents was found to increase with increasing driving pressure. Simulations using the Rayleigh, Plesset, Noltingk, Neppiras, Poritsky (RPNNP) equation were performed to study this behavior. Simulation results show that significant harmonic generation at high driving-pressure amplitudes contributes to the higher attenuation. Other possible explanations for this behavior were also examined. Attenuation coefficients of two contrast agents were also measured using a broadband method. The results showed great inconsistency when the center frequency of the incident broadband pulse was changed, indicating that broadband techniques may not be suitable for contrast-agent attenuation measurements.

Albumins↗