Cloning and identification of a novel thyroid hormone receptor ß isoform expressed in the pituitary gland.

We have previously identified a novel Trß isoform (TrßΔ) in the rat, in which a novel exon N (108 bps) was found between exon 3 and exon 4 of TrßΔ, which represents the only difference between TrßΔ and Trß1. In this study, we searched for an elongated Trß2-like subtype with one additional exon N. We successfully isolated the entire mRNA/cDNA of a novel elongated Trß2 isoform via PCR in the rat pituitary gland. The mRNA/cDNA was only 108 bps (exon N) longer than that Trß2, and the extension of the sequence was between exon 3 and 4 of Trß. The whole sequence of this novel Trß isoform has been published in NCBI GenBank (HM043807.1); it is named TRbeta2Delta (Trß2Δ). In adult rat pituitary tissue, quantitative real-time RT-PCR analysis showed that the mRNA levels of Trß2Δ and Trß2 were roughly equal (P > 0.05). We cloned, expressed, and purified the His-Trß2Δ protein [recombinant TRß2Δ (rTRß2Δ)]. SDS-PAGE and western blotting revealed that the molecular weight of rTRß2Δ was 58.2 kDa. Using a radioligand binding assay and an electrophoretic mobility shift assay, rTRß2Δ-bound T3 with high affinity and recognized thyroid hormone response element (TRE) binding sites. Finally, in vitro transfection experiments further confirmed that rTRß2Δ binding T3 significantly promotes the transcription of target genes via the TRE. Here, we have provided evidence suggesting that rTRß2Δ is a novel functional TR isoform.

[The cloning, expression and the binding ability with TRß1 of retinoid X receptor-α gene].

Retinoid X receptor-α (RXR-α), a member of nuclear receptor family, is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription. To further study the function of RXR-α, abundant of recombinant RXR-α protein in hand is necessary. In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α. Thereafter, competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium. Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses. The results showed that a protein, with the molecular mass around 50 kDa, could be selectively recognized by anti-RXR-α antibody. Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRß1 to form a heterodimer, which could specifically bind the target DNA fragment. This was confirmed by EMSA. In conclusion, the recombinant human retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.

[Interleukin-6 and tumor necrosis factor-alpha levels of endothelial cells in different hypoxia modes: in vitro experiment].

OBJECTIVE: To investigate the damage of different patterns of intermittent hypoxia (IH) and continuous hypoxia (CH) on endothelial cells. METHODS: Human umbilical vein endothelial cells of the line ECV304 were cultured in a program-controlled gas delivery system newly developed and divided into 8 groups to undergo different IH/reoxygenation (ROX) cycles so as to simulate the patterns of hypoxic episode seen in recurrent apnea and chronic obstructive pulmonary disease: intermittent normoxia (IN) group (exposed to 21% O2 15 s/21% O2 225 s for 60 cycles), IH group (exposed to 1.5% O2 15 s/21% O2 225 s for 30 or 60 cycles), IH hypercapnia group (exposed to 1.5% O2 and 20% CO2 15 s/21% O2 and 5% CO2 225 s, for 60 cycles), continuous hypoxia (CH group, exposed to 1.5% or 10% O2 for 15, 30 or 60 min), CH hypercapnia group (exposed to 10% O2 and 10% CO2 for 15, 30 or 60 min), CH added to IH group (exposed to 1.5% O2 15 s/10% O2 225 s for 60 cycles), different intermittent hypoxia extent group (exposed to 1.5% or 10% O2 15 s/21% O2 225 s for 60 cycles), different intermittent hypoxia frequency group (exposed to 1.5% O2 15 s/21% O2 225 s 315 s, 495 s or 105 s for 60 cycles), and different intermittent hypoxia duration group (exposed to 1.5% O2 15 s or 30 s/21% O2 225 s for 60 cycles). Then ELISA was conducted to examine the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha) Bicinchoninic acid method was used to standardize the cell total protein level. RESULTS: The levels of IL-6 and TNFalpha levels in the IH group were (770.40 +/- 21.60) and (126.93 +/- 2.58) pg.ml(-1).100 mg protein(-1) respectively, both significantly higher than those in the IN group [(374.06 +/- 38.10) and (31.96 +/- 13.64) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002], but significantly lower than those in the IH hypercapnia group [(829.27 +/- 7.16) and (78.77 +/- 4.00) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002]. The IL-6 levels of the CH hypercapnia 15, 30, and 60 min subgroups were all significantly higher than those of the corresponding CH subgroup (U = 0.000, P = 0.002). The IL-6 and TNFalpha levels of the CH added to IH group were (536.74 +/- 14.97) and (51.10 +/- 6.80) pg.ml(-1).100 mg protein(-1) respectively, both were significantly higher than those of the IN group, but significantly lower than those of the IH group (chi(2) = 23.4, P < 0.05). The levels of IL-6 and TNFalpha increased along with the increase of the IH degree (chi(2) = 23.4, P < 0.05). The level changes of IL-6 and TNFalpha of the groups with different intermittent hypoxia frequency and with different intermittent hypoxia duration were complicated. CONCLUSION: IH and CH significantly damage the endothelial cells dose-dependently, especially combined with hypercapnia. In IH/ROX, the inflammatory damage comes from ROX phase but not IH phase. Hypoxia duration and hypoxia frequency are also important parameters in the activation of inflammation.

[Changes of nuclear factor-kappaB and intercellular adhesion molecule-1 in endothelial cells exposed to various intermittent hypoxia].

OBJECTIVE: To construct a novel in vitro endothelial cell system to explore the changes of nuclear factor-kappaB (NF-kappaB) and intercellular adhesion molecule-1 (ICAM-1) levels after exposure to various intermittent hypoxia (IH) and re-oxygenation, an IH/reoxygenation (ROX) model. METHODS: We developed a gas control delivery system that permitted the exposure of ECV304 cell cultures, immortalized endothelial cell strain cultures of human umbilical vein endothelial cells (HUVEC), to IH/ROX cycles, simulating the pattern of hypoxic episodes seen in recurrent apnea. Cell samples were divided into the following groups according to IH duration/ROX duration. Group A (Intermittent Normoxia Group): 21% O(2) 15 s/21% O(2) 3 min 45 s; Group B (Standard Culture Group): no exposure; Group C: 1.5% O(2) 15 s/21% O(2) 3 min 45 s; Group D: 10% O(2) 15 s/21% O(2) 3 min 45 s; Fixed IH protocol as 1.5% O(2) 15s and ROX extent to 21% O(2), IH/ROX frequencies varied as 12 (Group C), 9.23 (Group E), 6.32 (Group F), 20 (Group G) and 40 (Group H) episodes per hour; Group I: 1.5% O(2) 30 s/21% O(2) 3 min 45 s; and after the exposure of Group C, the cell cultures were exposed to standard incubation device for 60 min (Group J) and 120 min (Group K). Prepared cell lysates and cell monolayers were analyzed for NF-kappaB levels and ICAM-1 levels in this IH model with enzyme-linked immunosorbent assay (ELISA) and cellular surface ELISA, and the cell total protein levels were measured with the method of bicinchoninic acid for standardization. SPSS 11.5 software package was used for statistical analysis. RESULTS: NF-kappaB and ICAM-1 levels in Group C were (0.82 +/- 0.28) and (1562 +/- 56) pg/ml, and those in Group A were (0.37 +/- 0.07) and (768 +/- 80) pg/ml, which showed statistical significance as compared with Group C (D = 225.00, 176.04, P < 0.01, < 0.05, respectively). Their levels in Group D were (0.66 +/- 0.22) and (1113 +/- 76) pg/ml, which were also significantly lower than Group C (U = 25.00, 0.00, all P < 0.01). NF-kappaB and ICAM-1 levels in Group I were (0.45 +/- 0.16) and (1155 +/- 19) pg/ml, which were statistically significant compared with Group C (U = 27.00, 0.00, all P < 0.01). In the same time, IH group had the relatively highest NF-kappaB and ICAM-1 levels amongst groups C, E, F, G and H. Which had different IH frequencies (chi(2) = 35.63, 56.89, all P < 0.01). NF-kappaB levels in Group J [(0.6233 +/- 0.0534)] did not differ significantly from Group C (D = 36.00, P > 0.05) and NF-kappaB levels in Group K [(0.3050 +/- 0.0013)] were lower than Group C (D = 234.00, P < 0.01). CONCLUSIONS: Our data indicated a selective and dose-dependent activation of inflammatory pathways on ECV304 cells by IH/ROX cycles with moderate frequencies, and a long time was needed for the cell rehabilitation from IH/ROX exposure.

Pre-deliver chitosanase to cells: a novel strategy to improve gene expression by endocellular degradation-induced vector unpacking.

A radio-labeled plasmid pTracer/Bsd/LacZ containing LacZ reporter gene was complexed with different molecular weights of chitosans (CS). Mouse myoblast cell line C2C12 was transfected by these chitosan-plasmid DNA complexes, and lipofectamine 2000 was used as control. Forty-eight hours after transfection, the activity of beta-galactosidase and radioactive count of cell lysis were determined. It was found that chitosan, especially low molecular weight species, had a surprising ability to deliver DNA into cells, since the radioactive count of cells transfected by chitosan-DNA complexes was even two times that of cells transfected by lipofectamine 2000. But the beta-galactosidase activity of chitosan/DNA complexes was much lower compared to that of lipofectamine 2000. Chitosanase which could degrade chitosan in specific mode was transported into C2C12 cells by osmotic lysis prior to gene delivery. Then these chitosanase-modified cells were transfected by CS-DNA complexes. The results indicated that beta-galactosidase activity in these cells increased markedly to 425.4 +/- 45.1 U/mg protein, nearly two-fold as that of cells transfected by liposome. This transfection protocol was also applied to 3T3 mouse fibroblast, 2T3 mouse osteoblast and MG63 human osteosarcoma cell lines, and an increased gene expression level was observed without exception. It is thought that the incorporated chitosanase could aid in chitosan degradation, which would promote gene unpacking, consequently increasing gene expression.

[Cloning and eukaryotic expression of wild type and truncated mouse ciliary neurotrophic factor gene].

OBJECTIVE: To clone and eukaryotic express wild type and truncated mouse ciliary neurotrophic factor (CNTF) gene, and to observe the biological effect of two types of CNTF gene expressing in ARPE-19 cells. METHODS: RT-PCR was used to amplify the cDNA of CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected to ARPE-19 cells. Dot blotting was used to detect the expression of CNTF. MTT and flow cytometry apoptosis assay were performed to observe the biological effect of CNTF expressing in ARPE-19 cells. RESULTS: Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF were detected in the supernatant of cells culture. MTT result shows that two types of CNTF have no proliferation promoting effect to ARPE-19 cells, and quantitive apoptosis assay implicated that CNTF could partially suppress the apoptosis that induced by the cells culturing with serum free culture. CONCLUSION: Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.

[Effects of various iodin-nutritional on activity of T4 5'-and 5-deiodinase in rat brain].

OBJECTIVE: To investigate the changing of T4 5'-and 5-deiodinase within rat brain under various iodin-nutritional states. METHODS: Animal model of iodine-deficiency rat was performed and the rats were divided into 4 groups by the intake of iodine-nutrition, and then killed at an age of 20 days. The thyroid hormones level in serum was measured by ELISA and the activity of T(4) 5'-and 5-deiodinase within brain was analyzed. RESULTS: In less-iodine (LI) group,TT4 and FT4 were accounting for 3.5% of the neutral-iodine (NI) group's, and FT3 was 174.0% of NI group's (P < 0.05). In NI group,TT4 and FT4 were 114.5% and 127.7% of NI group's (P < 0.05). In high-iodine (HI) group, TT4 and FT4 were 61.86% and 62.0% of NI group's, and FT3 was 184.9% of NI group's (P < 0.05). In LI group, the activity of T4 5'-deiodinase tissue of per gram (1.95 +/- 0.32) ngT3.microgT4(-1).h was significantly higher than that of NI group (P < 0.05), and the activity of 5-deiodinase (1.38 +/- 0.21) ngrT3.microg T4(-1).h(-1) is significantly less than that of NI group (1.59 +/- 0.23) (P < 0.05). In HI group the activity of T4 5'-and 5-deiodinase tissue of per gram (1.12 +/- 0.19 and 1.73 +/- 0.36) ngrT3.microgT4(-1).h(-1)was significantly less than that of NI group (P < 0.05). CONCLUSION: The activity of T4 5'-deiodinase in iodine deficiency heightens and that in iodine excess is debased, the activity of T4 5-deiodinase in iodine deficiency and in iodine excess is debased.

[Cloning and expression of an Aspergillus fumigatus chitosanase gene].

According to published DNA sequence of Aspergillus fumigatus chitosanase(Csn) gene, 8 long single DNA strands each about 100bp and 4 DNA primers were designed and synthesised. By PCR, 8 DNA strands were connected into a complete chitosanase gene of 624bp. This chitosanase gene was not identical with its wild type, some point mutations were introduced into its DNA sequence by special design of those 8 DNA strands. These mutaions did not change amino acid composition of the chitosanase, however, the codens were changed into E. coli favorites. The Csn gene was cloned into plasmid pGEM-Teasy and verified by DNA sequence analysis. Thereafter, Csn gene was subcloned into a fusion-protein expressing vector pGEX-3X. Recombinant plasmid pGEX-Csn was transformed into E. coli DH5alpha and the transformant was induced expressing with 0.5 mmol/L IPTG. Expressing product was analyzed by SDS-PAGE, fusion protein GST-Csn was purified by affinity chromatography. By factor X a digestion GST-Csn was cleaved and GST was taken out by another chromatography. The biological activity of recombinant chitosanase(rCsn) was also detected, as a result the recombinant Csn had a strong ability of degrading chitosan, which was much higher than lysozyme. Its chitosan-degradation activity could be influenced by pH and temperature.

[The cloning and expression of rat insulin-like growth factor I gene].

In this study an exons-connecting technique was used to amplify the complete DNA sequence encoding the mature peptide of IGF-I. Two pairs of primers were synthesized, primer a (Pa) and primer b (Pb) were used to amplify exon 2 coding fragment while primer c (Pc) and primer d (Pd) for amplifying exon 3 coding fragment. Pb was 40 bp, 18 bp at its 5' end was same with the anti-sense of exon 3's 5' end. Pc consisted of 41 bp with 22 bp at its 5' end consistent with the sense of the exon 2's 3' end. Genome DNA was extracted from rat liver. Using rat genome DNA as template PCR was performed with Pa, Pb and Pc, Pd as primers respectively. Two kinds of PCR products were obtained. One was 90 bp corresponding with the exon 2, another was 160 bp corresponding with the exon 3. Another PCR was done with these two PCR products used as not only template but also primers for each other. A 210 bp DNA fragment was produced, which encodes the mature peptide of rat IGF-I. Firstly the gene was cloned into plasmid pUC18. Then the recombinant plasmid pUC-IGF-I was digested with restriction endonuclease BamHI and EcoRI, plasmid pGEX-3X was digested with the same enzyme. IGF-I gene was linked into pGEX-3X and expressing plasmid pGEX-IGF-I was constructed. Plasmid pGEX-IGF-I was transformed into E. coli DH5 alpha and induced to express by IPTG. Expressed protein was analysized by SDS-PAGE. A fusion protein of GST-IGFI about 32.5 kDa could be observed. Western blot was performed with goat anti-human IGF-I IgG as primary antibody and donkey anti-goat IgG HRP as secondary antibody. Its result confirmed that the fusion protein really containing IGF-I. GST-IGFI was purified by affinity-chromatography and carrier-protein GST was cut off by Factor Xa. The bioactivity of purified IGF-I was detected with cultured NIH3T3 cell. The recombinant rat IGF-I can promote cell proliferation, shown by the elevation of 3H-TdR incorporation.

[The construction of yak liver cDNA library].

Yak is a very useful animal. The cDNA library of yak liver was constructed, which made a preparation for further cloning and expressing of wanted genes.mRNA from yak liver was extracted and purified, and cDNA was obtained by reverse transcription,and then cDNA library was constructed using plasmid pSPORT1 as the vector. The volume of the library was 1 x 10(6) transformants/microg cDNA and the rate of recombination was 93%. The results indicated that the cDNA library had enough volume for selecting wanted genes and made the basic of studying yak's genetic background.