AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method b...AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.展开更多
The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was ...The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.展开更多
Fourier transform infrared spectroscopy （FTIR） was employed to study the human epidermis larynx carcinoma cell lines （Hep-2） which were irradiated by different doses of X-ray. The results show that （1） the irrad...Fourier transform infrared spectroscopy （FTIR） was employed to study the human epidermis larynx carcinoma cell lines （Hep-2） which were irradiated by different doses of X-ray. The results show that （1） the irradiation of X-ray damages the structure of the CH3 groups of the thymine in DNA, which restrains the reproduction of Hep-2 cells effectively, （2） the 8 Gy dose of X-ray irradiation changes the framework and the relative contents of some proteins, lipids and the nucleic acid molecules intercellular in the greatest degree, and （3） the 8 Gy dose of X-ray irradiation is the best irradiation dose for lowering the degree of the cancerization of Hep-2 cells according to the criteria for the degree of the cancerization reported recently. Meanwhile, the apoptosis of these cells were detected by using flow cytometry （FCM） primarily. It shows that the apoptotic ratio of the Hep-2 cells depends on the irradiation dose to some extent, but is not linearly. And the apoptotic ratio of the 12 Gy dose group is the maximum （20.36%）, but the apoptotic ratios of the 2 to 8 Gy dose groups change little.展开更多
The Raman spectra from leukemic cell line （HL60） and normal human peripheral blood mononuclear cells （PBMCs） are obtained by confocal micro-Raman spectroscopy using near-infrared laser （785 nm） excitation. The s...The Raman spectra from leukemic cell line （HL60） and normal human peripheral blood mononuclear cells （PBMCs） are obtained by confocal micro-Raman spectroscopy using near-infrared laser （785 nm） excitation. The scanning range is from 500 to 2000 cm^-1. The two average Raman spectra of normal PBMCs and carcinoma cells have clear differences because their structure and amount of nucleic acid, protein, and other major molecules are changed. The spectra are also compared and analyzed by principal component analysis （PCA） to demonstrate the two distinct clusters of normal and transformed cells. The sensitivity of this technique for identifying transformed cells is 100%.展开更多
基金the Natural Scientific Foundation of China (NSFC3962526)National High-Technology Project-863 (102-10-01-04)
文摘AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.
基金This work was supported by a grant from 863High Technology Program,Chinese Ministry of Sci-ence and Technology
文摘The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.
基金the Science and Technol-ogy Department of Henan province (No.0624410052)the key Ion Beam Bioengineering Laboratory of Henan Province+1 种基金the Pathology Laboratory of Zhengzhou Uni-versitythe People's Hospital of Henan Province.
文摘Fourier transform infrared spectroscopy （FTIR） was employed to study the human epidermis larynx carcinoma cell lines （Hep-2） which were irradiated by different doses of X-ray. The results show that （1） the irradiation of X-ray damages the structure of the CH3 groups of the thymine in DNA, which restrains the reproduction of Hep-2 cells effectively, （2） the 8 Gy dose of X-ray irradiation changes the framework and the relative contents of some proteins, lipids and the nucleic acid molecules intercellular in the greatest degree, and （3） the 8 Gy dose of X-ray irradiation is the best irradiation dose for lowering the degree of the cancerization of Hep-2 cells according to the criteria for the degree of the cancerization reported recently. Meanwhile, the apoptosis of these cells were detected by using flow cytometry （FCM） primarily. It shows that the apoptotic ratio of the Hep-2 cells depends on the irradiation dose to some extent, but is not linearly. And the apoptotic ratio of the 12 Gy dose group is the maximum （20.36%）, but the apoptotic ratios of the 2 to 8 Gy dose groups change little.
基金supported by the National Natural Science Foundation of China(No.60778046,60711120031)the Natural Science Foundation of Fujian Province(No.A0740010,2007J0318)
文摘The Raman spectra from leukemic cell line （HL60） and normal human peripheral blood mononuclear cells （PBMCs） are obtained by confocal micro-Raman spectroscopy using near-infrared laser （785 nm） excitation. The scanning range is from 500 to 2000 cm^-1. The two average Raman spectra of normal PBMCs and carcinoma cells have clear differences because their structure and amount of nucleic acid, protein, and other major molecules are changed. The spectra are also compared and analyzed by principal component analysis （PCA） to demonstrate the two distinct clusters of normal and transformed cells. The sensitivity of this technique for identifying transformed cells is 100%.
