Publications by authors named "Yong-Jun Xu"

17 Publications

  • Page 1 of 1

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats.

J Neuroinflammation 2021 Apr 2;18(1):85. Epub 2021 Apr 2.

Department of Gynecology and Obstetrics and Research Center for Molecular Metabolomics, Xiangya Hospital Central South University, Changsha, 410008, China.

Background: Prenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure.

Methods: Pregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro.

Results: Both male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment.

Conclusion: CRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.
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http://dx.doi.org/10.1186/s12974-021-02129-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019183PMC
April 2021

Verification on the Developmental Toxicity of Short-term Exposure to Phenol in Rats.

Biomed Environ Sci 2020 Jun;33(6):403-413

China CDC Key Laboratory of Environment and Population Health, National Institute of Environmental Health, Chinese Center for Disease Control and Prevention, Beijing 100021, China.

Objective: To verify the health advisory for short-term exposure to phenol.

Methods: The method of this validation experiment was the same as the US Environmental Protection Agency (EPA) methodology for toxicology experiments used to determine phenol drinking water equivalent level (DWEL). Pregnant female Sprague-Dawley rats were administered phenol in distilled water by gavage at daily doses of 15, 30, 60, 120, and 240 mg/kg body weight (b.w.) from implantation (the 6th day post-mating) to the day prior to the scheduled caesarean section (the 20th day of pregnancy). The following information was recorded: general behavior; body weight; number of corpus luteum, live birth, fetus, stillbirth, and implantation; fetal gender; body weight; body length; tail length; and abnormalities and pathomorphological changes in the dams.

Results: In the 60 mg/kg b.w. dose group, the mortality of pregnant rats increased with increasing doses, suggesting maternal toxicity. Fetal and placental weights decreased as phenol dose increased from 30 mg/kg b.w., and were significantly different compared those in the vehicle control group, which suggested developmental toxicity in the fetuses. However, the phenol-exposed groups showed no significant change in other parameters compared with the vehicle control group ( > 0.05).

Conclusion: Despite using the same method as the US EPA, a different NOEAL of 15 mg/(kg·d) was obtained in this study.
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http://dx.doi.org/10.3967/bes2020.055DOI Listing
June 2020

Further identification of a 140bp sequence from amid intron 9 of human FMR1 gene as a new exon.

BMC Genet 2020 06 18;21(1):63. Epub 2020 Jun 18.

Department of Clinical Genetics and Experimental Medicine, 900th Hospital of the Joint Logistics Force, Xiamen University School of Medicine, 156 Xi'erhuanbei Road, Fuzhou City, Fujian Province, 350025, People's Republic of China.

Background: The disease gene of fragile X syndrome, FMR1 gene, encodes fragile X mental retardation protein (FMRP). The alternative splicing (AS) of FMR1 can affect the structure and function of FMRP. However, the biological functions of alternatively spliced isoforms remain elusive. In a previous study, we identified a new 140bp exon from the intron 9 of human FMR1 gene. In this study, we further examined the biological functions of this new exon and its underlying signaling pathways.

Results: qRT-PCR results showed that this novel exon is commonly expressed in the peripheral blood of normal individuals. Comparative genomics showed that sequences paralogous to the 140 bp sequence only exist in the genomes of primates. To explore the biological functions of the new transcript, we constructed recombinant eukaryotic expression vectors and lentiviral overexpression vectors. Results showed that the spliced transcript encoded a truncated protein which was expressed mainly in the cell nucleus. Additionally, several genes, including the BEX1 gene involved in mGluR-LTP or mGluR-LTD signaling pathways were significantly influenced when the truncated FMRP was overexpressed.

Conclusions: our work identified a new exon from amid intron 9 of human FMR1 gene with wide expression in normal healthy individuals, which emphasizes the notion that the AS of FMR1 gene is complex and may in a large part account for the multiple functions of FMRP.
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http://dx.doi.org/10.1186/s12863-020-00870-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301526PMC
June 2020

Splicing of exon 9a in FMR1 transcripts results in a truncated FMRP with altered subcellular distribution.

Gene 2020 Mar 11;731:144359. Epub 2020 Jan 11.

Department of Clinical Genetics and Experimental Medicine, 900th Hospital of the Joint Logistics Force, Fujian Medical University, Fuzhou, Fujian 350025, China. Electronic address:

FMRP is an RNA-binding protein, loss of which causes fragile X syndrome (FXS). FMRP has several isoforms resulted from alternative splicing (AS) of fragile X mental retardation 1 (FMR1) gene, but their biological functions are still poorly understood. In the analysis of alternatively spliced FMR1 transcripts in the blood cells from a patient with FXS-like phenotypes (normal CGG repeats and no mutation in coding sequence of FMR1), we identified three novel FMR1 transcripts that include a previously unidentified microexon (46 bp), terming the exon 9a. This microexon exists widely in unaffected individuals, inclusion of which introduces an in-frame termination codon. To address whether these exon 9a-containing transcripts could produce protein by evading nonsense-mediated decay (NMD), Western blot was used to analysis blood cell lysate from unaffected individuals and a 34 kDa protein that consistent in size with the molecular weight of the predicted truncated protein produced from mRNA with this microexon was found. Meanwhile, treatment of peripheral blood mononuclear cells with an inhibitor of NMD (Cycloheximide) did not result in significant increase in exon 9a-containing transcripts. Using confocal immunofluorescence, we found the truncated protein displayed both nuclear and cytoplasmic localization in HEK293T and HeLa cells due to lacking C-terminal domains including KH2, NES, and RGG, while the full-length FMRP protein mainly localized in the cytoplasm. Therefore, we hypothesize that the inclusion of this microexon to generate exon 9a-containing transcripts may regulate the normal functionality of FMRP, and the dysregulation of normal FMRP due to increased exon 9a-containing alternatively spliced transcripts in that patient may be associated with the manifestation of FXS phenotype.
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http://dx.doi.org/10.1016/j.gene.2020.144359DOI Listing
March 2020

CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.

FASEB J 2018 08 15;32(8):4258-4269. Epub 2018 Mar 15.

Department of Physiology, Second Military Medical University, Shanghai, China.

Pregnant women at risk of preterm labor usually receive synthetic glucocorticoids (sGCs) to promote fetal lung development. Emerging evidence indicates that antenatal sGC increases the risk of affective disorders in offspring. Data from animal studies show that such disorders can be transmitted to the second generation. However, the molecular mechanisms underlying the intergenerational effects of prenatal sGC remain largely unknown. Here we show that prenatal dexamethasone (Dex) administration in late pregnancy induced depression-like behavior in first-generation (F1) offspring, which could be transmitted to second-generation (F2) offspring with maternal dependence. Moreover, corticotropin-releasing hormone (CRH) and CRH receptor type 1 (CRHR1) expression in the hippocampus was increased in F1 Dex offspring and F2 offspring from F1 Dex female rats. Administration of a CRHR1 antagonist to newborn F1 Dex offspring alleviated depression-like behavior in these rats at adult. Furthermore, we demonstrated that increased CRHR1 expression in F1 and F2 offspring was associated with hypomethylation of CpG islands in Crhr1 promoter. Our results revealed that prenatal sGC exposure could program Crh and Crhr1 gene expression in hippocampus across 2 generations, thereby leading to depression-like behavior. Our study indicates that prenatal sGC can cause epigenetic instability, which increases the risk of disease development in the offspring's later life.-Xu, Y.-J., Sheng, H., Wu, T.-W., Bao, Q.-Y., Zheng, Y., Zhang, Y.-M., Gong, Y.-X., Lu, J.-Q., You, Z.-D., Xia, Y., Ni, X. CRH/CRHR1 mediates prenatal synthetic glucocorticoid programming of depression-like behavior across 2 generations.
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http://dx.doi.org/10.1096/fj.201700948RRDOI Listing
August 2018

Discovery of 2-((3-cyanopyridin-2-yl)thio)acetamides as human lactate dehydrogenase A inhibitors to reduce the growth of MG-63 osteosarcoma cells: Virtual screening and biological validation.

Bioorg Med Chem Lett 2016 08 30;26(16):3984-7. Epub 2016 Jun 30.

Department of Orthopedics, Affiliated Hospital of Putian University, Putian 351100, Fujian Province, China. Electronic address:

Lactate dehydrogenase A (LDHA) has emerged as an attractive target in the oncology field. In this paper, we present the identification of 2-((3-cyanopyridin-2-yl)thio)acetamide-containing compounds as LDHA inhibitors. The in vitro enzymatic assay suggested that inhibitor 9 had good inhibitory potency against LDHA with IC50 value as 1.24μM. Cytotoxicity assay showed that inhibitor 9 strongly inhibited the proliferation of cancer cell MG-63 (EC50=0.98μM). These findings indicated that inhibitor 9 could be employed as a lead for developing more potent LDHA inhibitor with anti-proliferative potency.
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http://dx.doi.org/10.1016/j.bmcl.2016.06.083DOI Listing
August 2016

meso-Tetrakis[4-(heptyloxy)phenyl]porphyrin.

Acta Crystallogr C 2013 Jun 15;69(Pt 6):651-3. Epub 2013 May 15.

College of Chemistry and Environmental Engineering, Dongguan University of Technology, Guangdong 523808, People's Republic of China.

The core of the novel title centrosymmetric porphyrin derivative, C72H86N4O4, with long flexible hexyloxy substituents, is almost planar, which is anticipated to facilitate π-electron delocalization and lead to a significant deviation between the planes of the benzene rings and the molecular plane. The two N-bound H atoms on the pyrrole rings are disordered and the occupancy factors refined to a ratio of 0.28 (2):0.72 (2).
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http://dx.doi.org/10.1107/S0108270113011232DOI Listing
June 2013

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

Zhonghua Nan Ke Xue 2009 Nov;15(11):980-4

Research Center of Reproductive Medicine, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, China.

Objective: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility.

Methods: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy.

Results: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01).

Conclusion: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.
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November 2009

3-Hydr-oxy-1,2-dimethoxy-anthraquinone.

Acta Crystallogr Sect E Struct Rep Online 2009 Jun 6;65(Pt 7):o1524. Epub 2009 Jun 6.

College of Chemistry and Environmental Engineering, Dongguan University of Technology, Dongguan 523808, People's Republic of China.

The title compound, C(16)H(12)O(5), was isolated from Morinda officinalis How. The anthraquinone ring system is almost planar, the dihedral angle between the two benzene rings being 1.12 (4)°. In the crystal structure, O-H⋯O and C-H⋯O hydrogen bonds link the mol-eculesin the crystallographic a-axis direction. Weak π-π stacking inter-actions [centroid-centroid distance between symmetry-related benzene rings of 3.699 (4) Å] are also present.
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http://dx.doi.org/10.1107/S1600536809021266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2969515PMC
June 2009

Poly[diaqua-(μ-oxalato)(μ-2-oxido-pyridinium-3-carboxyl-ato)praseo-dymium(III)].

Acta Crystallogr Sect E Struct Rep Online 2009 Feb 21;65(Pt 3):m310. Epub 2009 Feb 21.

College of Chemistry and Environmental Engineering, Dongguan University of Technology, Dongguan 523808, Guangdong, People's Republic of China.

In the title complex, [Pr(C(6)H(4)NO(3))(C(2)O(4))(H(2)O)(2)](n), each Pr(III) ion is coordinated by eight O atoms from two 2-oxynicotinate ligands, two oxalate ligands and two water mol-ecules, displaying a distorted bicapped square-anti-prismatic geometry. The carboxyl-ate groups link adjacent praseodymium metal centres, forming layers parallel to the bc plane. The crystal packing is stabilized by inter-molecular O-H⋯O and N-H⋯O hydrogen bonds.
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http://dx.doi.org/10.1107/S160053680900542XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2968694PMC
February 2009

Degradation of acenaphthene by ozone.

Biomed Environ Sci 2007 Aug;20(4):291-4

Laboratory of Environmental Technology, INET Tsinghua University, Beijing 100084, China.

Objective: To investigate the oxidation of acenaphthene (Ace), a polycyclic aromatic hydrocarbon (PAH) with a saturated C-C bond by ozone and to characterize the intermediate products of ozonation.

Methods: Ozone was generated from filtered dry oxygen by an ozone generator and continually bubbled into a reactor containing 1g/L Ace dissolved in an acetonitrile/water solvent mixture (90/10, v/v) at a rate of 0.5 mg/s. HPLC was used to analyze the Ace concentration. Total organic carbon (TOC) was used to measure the amount of water soluble organic compounds. GC-MS was used to identify the ozonized products. Oxygen uptake rate (OUR) of activated sludge was used to characterize the biodegradability of ozonized products.

Results: During the ozonation process, Ace was degraded, new organic compounds were produced and these intermediate products were difficult mineralize by ozone, with increasing TOC of soluble organics. The ozonized products were degraded by activated sludge more easily than Ace.

Conclusion: Ozonation decomposes the Ace and improves its biodegradability. The ozonation combined with biological treatment is probably an efficient and economical way to mineralize acenaphthene in wastewater.
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August 2007

[Radioligand binding assay of progesterone receptors on normal fertile human sperm membrane].

Zhonghua Nan Ke Xue 2007 Feb;13(2):114-7

Research Center of Reproductive Medicine, Xi'an Jiaotong University School of Medicine, Key Laboratory of Environment and Diseases-related Genes, Ministry of Education, Xi'an, Shaanxi 710061, China.

Objective: To investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation.

Methods: Viable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression.

Results: Kd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01.

Conclusion: There is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.
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February 2007

[Study on detection method of 8-OH-dG in DNA extracted from HepG2 cells in vitro by capillary zone electrophoresis].

Wei Sheng Yan Jiu 2005 Sep;34(5):539-42

Institute for Environmental Hygiene and Health-Related Product Safety, Chinese Center for Disease Control and Prevention, Beijing 100021, China.

Objective: To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis.

Methods: HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation. 0 and 15 mmol/L H2O2 were added into two groups of HepG2 cells (5 x 10(7)) respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37 degrees C with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis.

Results: The optimized conditions were: uncoated silica capillary (47 cm x 50 microm i.d.), 20 mmol/L borate buffer (pH 9.5), 25 degrees C, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2-untreated DNA increased.

Conclusion: The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.
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September 2005

Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals.

World J Gastroenterol 2003 Jul;9(7):1460-4

Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China.

Aim: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs.

Methods: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL(3)-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG(2), both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG(2) cells stably transfected by the recombinant vector (HepG(2)-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG(2) cells (HepG(2)-wt).

Results: The inducible luciferase expression of HepG(2)-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG(2)-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG(2)-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG(2)-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 x 10(-12) to 5 x 10(-9) mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG(2)-luc and HepG(2)-wt cells. The correlation between TCDD doses from 1.1 x 10(-13) to 1 x 10(-8) mol/L and luciferase activities was also found to be significant in HepG(2)-luc cells (r=0.997, P<0.001), but not in their HepG(2)-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG(2)-luc cells were both better than that of EROD in HepG(2)-wt cells, the former was at 1.1 x 10(-13) mol/L and 3.5 x 10(-12) mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively.

Conclusion: The luciferase expression of HepG(2)-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615482PMC
http://dx.doi.org/10.3748/wjg.v9.i7.1460DOI Listing
July 2003

[Investigation of progesterone receptor on human sperm plasma membrane].

Zhonghua Nan Ke Xue 2002 ;8(4):277-80

Department of Histology and Embryology, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, China.

Objectives: To investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface.

Methods: After in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC).

Results: The spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%.

Conclusions: There is selective expression of PR on the human sperm acrosome surface.
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January 2003

Bioluminescent method for detecting telomerase activity.

Clin Chem 2002 Jul;48(7):1016-20

National Laboratory of Biomedical Photonics, Institute of Environmental Medicine, Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan 430030, The People's Republic of China.

Background: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA).

Methods: Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy.

Results: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively.

Conclusion: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.
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July 2002

Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals.

Biomed Environ Sci 2002 Mar;15(1):58-66

Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

Objective: To improve the chemically-activated luciferase expression (CALUX) bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs.

Methods: A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2 used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay.

Results: The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.11 pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100 pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.

Conclusion: The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.
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March 2002