Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.

Anti-inflammatory or proinflammatory effect of an adenosine receptor agonist on the Th17 autoimmune response is inflammatory environment-dependent.

Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1-type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoid-binding protein peptides 1-20. We showed that injection of mice with a nonselective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by γδ T cells, and that the response of γδ T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better AR-targeted therapies.

IL-23 receptor expression on γδ T cells correlates with their enhancing or suppressive effects on autoreactive T cells in experimental autoimmune uveitis.

We have previously reported that, depending on their activation status, mouse γδ T cells can either enhance or inhibit the activity of IL-17(+) autoreactive T cells in experimental autoimmune uveitis. In this study, we showed that γδ T cells in naive C57BL/6 (B6) mouse do not express the IL-23R, whereas in immunized mice, it is expressed on >50% of γδ T cells. In vitro studies showed that IL-23R expression on γδ T cells is modulated by their state of activation, as weakly activated γδ T cells expressed the IL-23R, but highly activated γδ T cells did not. Functional studies showed that IL-23R(+) γδ T cells had the strongest suppressive effect on IL-17(+) autoreactive T cells, and that this effect was inhibited when the IL-23R was blocked by anti-IL-23R Ab or in the presence of excessive amounts of exogenous IL-23. We conclude that the balance between the enhancing and inhibitory effects of γδ T cells is regulated by their level of IL-23R expression. The expression of variable IL-23R levels allows γδ T cells to have different regulatory effects on adaptive immune responses, conceivably as a result of αß and γδ T cells competing for IL-23.

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.

Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis.

In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αßTCR(+) regulatory T cells.

[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.

Improved transfection efficiency of CS/DNA complex by co-transfected chitosanase gene.

Previously, we had demonstrated that insufficient intracellular unpacking of exogene from its chitosan carrier contributes towards the restricted transfection efficiency of CS/DNA complex. In order to enhance intracellular unpacking and thus improve the transfection efficiency, our present work has addressed a novel strategy of chitosanase gene (csn) co-transfection. An Aspergillus fumigatuscsn gene was semi-synthesized and cloned into a prokaryotic expression vector, plasmid pGEX-3X, meanwhile a mutant csn gene encoding an inactive Asp129-Asn chitosanase was generated by site-directed mutagenesis. Both active csn (acCSN) and inactive csn (inCSN) genes were expressed in bacteria cells and chitosan degradation activities of those purified recombinant proteins were tested. These csn genes were further subcloned into an eukaryotic expression vector, plasmid pTracer-CMV/Bsd, containing a gfp reporter gene. Recombinant plasmid pTracer-accsn or pTracer-incsn was co-transfected with plasmid pTracer/Bsd/LacZ, which contains an additional lacZ reporter gene, into C2C12 myoblast cells by CS/DNA complex. The expression of gfp reporter gene was determined by fluorescence microscope, while the expression of lacZ reporter was evaluated quantitatively by beta-galactosidase activity. All together, findings indicate that during the exogene being delivered into mammalian cells by CS/DNA complex, the csn co-transfection is beneficial for the exogene expression.

[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.

Effect of iodine supplement on iodine status and 5'-deiodinase activity in the brain of neonatal rats with iodine deficiency.

The weanling Wistar rats of iodine deficiency were divided into three groups for supplementation of different levels of iodine (iodine-excessive [IE], iodine-adequate [IA], and iodine-deficient [ID]), with a control group (C). The iodine content in the thyroid was determined by epithermal neutron activation analysis. The activities of 5'-deiodinase and 5-deiodinase in the brains were assayed by determining the conversion ratios of T4 to T3 and rT3, respectively. The thyroid hormones levels in serum were also tested. The results indicated that the ID group had a goiter containing a small amount of iodine, but the IE group had a slightly swollen thyroid with rich iodine; the concentration of iodine per unit mass of thyroid was lower in group IE than in groups IA and C. The highest 5'-deiodinase and lowest 5-deiodinase activities in group ID and the lowest 5'-deiodinase activity in group IE were found. The iodine deficiency or excess resulted in a compensated hypothyroid state. The results suggest that the iodine status and the deiodinases activities would become normal for the rats of iodine deficiency if adequate iodine is supplemented soon after birth. Meanwhile, it is also critical to avoid excessive intake of iodine to reduce the risk for overcorrecting.

[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.

CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.

Correction: CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

[This corrects the article DOI: 10.1371/journal.pone.0150078.].

[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.

An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation.

We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c(+)Gr-1(+) dendritic cell subset that promotes the Th17 response.

Adenosine is one of the major molecules associated with inflammation. We have previously reported that an adenosine receptor (AR) agonist has an enhancing effect on Th17 autoimmune responses, even though it suppressed Th1 responses. To determine the mechanism involved, we have examined the effect of AR agonists on mouse bone marrow dendritic cell (BMDC) differentiation and function. We show that mouse bone marrow cells (BMCs) differentiated into CD11c(+)Gr-1(+) dentritic cells (DCs) when cultured in granulocyte macrophage colony-stimulating factor (GM-CSF)-containing medium containing an AR agonist. The non-selective AR agonist NECA and an A2BR-specific agonist had a similar effect, and the effect of NECA could be blocked by an A2BR-specific antagonist. Unlike CD11c(+)Gr-1(-) BMDCs, which have a greater stimulatory effect on Th1 T cells than Th17 cells, CD11c(+)Gr-1(+) BMDCs had a greater stimulatory effect on Th17 autoreactive T cells than on Th1 autoreactive T cells and this effect depended on γδ T cell activation.

Variations of elemental distribution in brain regions of neonatal rats at different iodine intakes.

Iodine deficiency (ID) can result in irreversible damage to the brain during the fetal and neonatal stages. As the active center of many enzymes, trace elements play essential roles in brain function. In this work, the relative contents and distributions of elements (Cl, Br, Fe, Cu, and Zn) in two important brain regions, the cerebral cortex and hippocampus, of the iodine-deficient model rats were determined by the synchrotron radiation X-ray fluorescence (SRXRF) method. Meanwhile, the ID rats were supplemented with adequate and excessive iodine, respectively. The results indicated that the distributions of trace elements could be influenced by the different iodine intakes in the stage of brain development. In contrast to the control group, the contents of Cl, Br, and Zn in two brain regions showed a significant increase in the ID group; however, both Fe and Cu decreased in the cerebral cortex and increased in the hippocampus in the ID group. In addition, only slight changes of elemental contents in brain were found between the adequate and excessive iodine-supplemented rats.

[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.