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Objective:To study the dynamic changes of the localization and expression of nuclear transporter KPNA2 in the occurrence and development of bronchopulmonary dysplasia(BPD) in neonatal rats, and explore its role in the pathogenesis of BPD in premature infants.Methods:The BPD model of newborn SD rats was induced with 85% oxygen concentration( n=50), and the control group was inhaled with air( n=50). The lung tissue samples were collected on 1 d, 3 d, 7 d, 10 d, and 14 d, respectively, in the two groups and separated.Purification and culture of alveolar type Ⅱ epithelial cells.The distribution and expression of KPNA2 were detected by immunohistochemistry, immunofluorescence, western blot and RT-PCR. Results:Immunohistochemistry showed that KPNA2 mainly located in alveolar epithelial cells′ cytoplasm and nucleus, and BPD group was more expressive than control group.Cell immunofluorescence showed that KPNA2 in control group was mainly localized in the nucleus, and in BPD group, KPNA2 was mainly localized in the cytoplasm from 3 d to 14 d. The nuclear expression of KPNA2 was weaker than that in the control group, and the cytoplasmic expression was stronger than that in the control group.The expression trends of KPNA2 total protein, plasma protein and mRNA were basically the same.The BPD group began to increase on the 1st day ( P<0.05), and was still higher than the control group on the 14th day( P<0.05); in BPD group, KPNA2 nucleoprotein expression began to decrease on the 3rd day( P<0.01), continued to decrease to 14 days( P<0.05). Conclusion:The dysfunction of KPNA2 nuclear transport in neonatal rats exposed to hyperoxia may be an important mechanism that affects the early initiation of the DNA damage response of BPD alveolar epithelial cells.
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Objective:To study the structure of the human THOC1-THOC2-THOC3 subcomplex by negative-staining electron microscopy. Methods:The recombinant protein components were co-expressed in cultured insect cells, and the protein complex was isolated via sequential Ni-NTA and Strep-Tactin affinity purification followed by Superose gel filtration chromatography. The purified protein sample was then subjected to negative-staining electron microscopy and single particle image analysis. Results:The recombinant human THOC1-THOC2-THOC3 complex with high purity and good homogeneity was obtained by using a tandem affinity purification scheme with Ni-NTA and streptavidin-based chromatography. Preliminary study on the structure of human THOC1-THOC2-THOC3 subcomplex was achieved by electron microscopy using negative staining with uranium formate. Conclusion:A low resolution model of human THOC1-THOC2-THOC3 subcomplex was achieved by single particle reconstruction.
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Transportation between the cytoplasm and the nucleoplasm is critical for many physiological and pathophysiological processes including gene expression, signal transduction, and oncogenesis. So, the molecular mechanism for the transportation needs to be studied not only to understand cell physiological processes but also to develop new diagnostic and therapeutic targets. Recent progress in the research of the nuclear transportation (import and export) via nuclear pore complex and four important factors affecting nuclear transport (nucleoporins, Ran, karyopherins, and nuclear localization signals/nuclear export signals) will be discussed. Moreover, the clinical significance of nuclear transport and its application will be reviewed. This review will provide some critical insight for the molecular design of therapeutics which need to be targeted inside the nucleus.
Assuntos
Transporte Ativo do Núcleo Celular , Carcinogênese , Fenômenos Fisiológicos Celulares , Citoplasma , Expressão Gênica , Carioferinas , Sinais de Localização Nuclear , Poro Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Transdução de Sinais , Meios de TransporteRESUMO
Objective To observe the influence of trastuzumab on DNA break repair and Her-2 nuclear import after radiation in breast cancer cell line SKBR3,and discuss the radiosensitivity mechanism of trastuzumab.Methods Clone formation assay was used to analyze the difference of survival fractions between radiation group and radiation plus trastuzumab group.Confocal microscopy was applied to observe the influence of trastuzumab in the nuclear import process of Her-2 and the expression of γH2AX after radiation,which is considered as the marker of DNA double strand break.Western blotting was used to detect the expression of Her-2 and DNA-PKcs in nuclei after radiation.Results The result of clone formation assayshowed that the SF2 in radiation group was 0.547±0.046 and 0.321±0.022 in the radiation plus trastuzumab group were significantly decreased,the results of confocal microscopy showed that trastuzumab postponed the nuclear import process of Her-2 (52.80±19.74 in radiation group,21.41±10.55 in the radiation group),and increased expression of γH2AX after radiation (85.40±25.63 in radiation group,18.53±44.32 in the radiation group),and western blotting revealed trastuzumab reduced the expression of Her-2,DNA-PKcs in nuclei.Conclusion Trastuzumab can inhibit the radiation induced nuclear import of Her-2,and decrease Her-2,DNA-PKcs in nuclei to increase the DSB on early stage after radiation.