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ObjectiveTo explore the clinical manifestations of infant cytomegalovirus infection in different age. MethodThe clinical data of 237 infants who suffered from cytomegalovirus infection was analyzed retrospectively and divided into three groups: 0-3 months old (77 cases,group A) ,4-6 months old ( 65 cases , group B ) , 7-12 months old ( 95 cases , group C ). ResultsThe incidence of respiratory infection was the highest among all infectious organs in three groups, the numbers of patients who had wheeze in group A was less than that in the other two groups[24.7％(19/77) vs. 61.5％ (40/65), 61.1％( 58/95 )](P ＜ 0.01 ).The incidence of jaundice decreased gradually as the babies grew up[23.4％( 18/77 )→7.7％(5/65 )→1.1％( 1/95 )](P ＜ 0.05). There was no significant difference in diarrhoea and bleeding among three groups (P＞0.05 ). The proportion of alanine aminotransferase], aspartate amino transferase increasing was similar among three groups, but gamma-glutamyl transferase (GGT) was different, the proportion of GGT increasing was 77.9％ (60/77) in group A which was higher than that in the other two groups[10.8％ ( 7/65 ), 2.1％ ( 2/95 )].Granulocytopenia in group B was obviously decreased compared with the other two groups (P ＜ 0.05 ), anemia was easily occurred in group C (P ＜ 0.05 ). ConclusionThe injury of cytomegalovirus infection may be related to month old.
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Purpose To investigate the effect of small interfering RNA on the increase of myocyte enhancer factor 2A(MEF2A) expression in PC12 cells exposed to hypoxia.Methods PC12 cells were cultured under normal conditions or were exposed to hypoxic conditions.Small interfering RNA targeted MEF2A gene (MEF2A-siRNA) was chemically synthesized. Eukaryocytic expression vector was built and transfected into PC12 cells with liposome. The expression of MEF2A mRNA was detected by real-time PCR. Western blot was used to detect the MEF2A protein.Results Compared with normal control(2~(-△△CT)=1.01±0.02), the mRNA level of MEF2A gene in PC12 cells with the treatment of MEF2A-siRNA was down-regulated significantly by 88%(2~(-△△CT)=0.12±0.03, P＜0.01).The expression of MEF2A protein in hypoxia-treated PC12 cells was markedly higher than that of normal control(98.4±11.7 and 47.5±7.6,P＜0.01).However, MEF2A-siRNA could significantly suppress the increase of MEF2A protein (P＜0.01).Conclusion MEF2A gene silence induced by siRNA might inhibit the increase of MEF2A protein by hypoxia in PC12 cells.
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Objective To identify the important regulatory elements in the promoter of human CD2 associated protein(CD2AP) by conserved sequence analysis among different species and luciferase functional detection. Methods The promoter sequences of CD2AP from different species were analyzed by BLAST. Plasmids containing different length of deletion mutations of human CD2AP promoter were constructed. Pro-moter activities were tested in 3 kinds of cells from different species by luciferase analysis and were tested in HEK-293 cells treated with all-trans-retinoic acid. Results Homologous sequence comparison in CD2AP promoter area among human, cattle and pig showed that putative specific protein 1 (Sp1) sites and down-stream promoter element (DPE) were highly evolutional]y conserved. Progressive deletion luciferase analysis of DNA fragments revealed similar promoter activity style among 3 different cell lines from 3 different spe-cies, HEK-293, BHK-21 and Vero cells. One basic promoter activity located within 500 bp upstream of ATG. Fragments of further upstream 100 bp or more had drastically 10 times increased promoter activity. Two putative Sp1 sites were in this 100 bp region. All-trans-retinoic acid decreased the luciferase activity of CD2AP promoter. Conclusion Putative Spl sites and DPE have important functions in the promoter activity of CD2AP.
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AIM: To investigate the effect of IL-10 on IL-1?-induced prostaglandin E 2(PGE 2) release and cyclooxygenase-2(COX-2) expression in human mesangial cells and to examine whether IL-10 has effect on the biological function of IL-1?.METHODS: The PGE 2 concentration in supernatants of HMC was measured by radioimmunoassay. The COX-2 mRNA and protein were measured by RT-PCR and Western blot, respectively. RESULTS: PGE 2 and COX-2 were significantly increased after treatment with IL-1?( P0.05 , respectively), while it inhibited IL-1?-elicited PGE 2 production, as well as COX-2 mRNA and protein expression in a concentration-dependent fashion. CONCLUSIONS: These results indicated that IL-10 depressed the IL-1?-induced release of PGE 2 and expression of COX-2. These data suggested that IL-10 could exert anti-inflammatory actions at several levels, not only by inhibiting the production of pro-inflammatory cytokines but also by suppressing their biological function.
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Objective To explore the inhibitory effect of mutant influenza A viruses to the activation of interferon regulatory factor 3 (IRF-3). Methods HEK293 cells were infected with A/FM/1/47,A/HK/1/68, A/HK/1/68-MA20, A/HK/1/68-MA20C and positive control Sendai virus (SV). Whether the slowly moved phosphorylation form Ⅲ and Ⅳ of IRF-3 appeared or not was compared by Western blot in cells infected with these viruses. Wild type of NS1 from A/HK/1/68 and mutant NS1 from A/HK/1/68-MA20 were subcloned into pcDNA3.1-flag respectively. They were transfected in HEK 293 cells respectively. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Whole cell extracts were analyzed by Western blot and then probed with monoclonal flag antibody to check the expression of NS1, or with anti-IRF-3 to observe the inhibitory effects of the wild and mutant NS1 to the activated IRF-3. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and wild or mutant NS1 cDNA expression plasmid. SV was used to infect these cells after the co-transfection. Results Only less virulent A/HK/1/68-MA20 and positive control SV can activate IRF-3. Activated form Ⅲ and Ⅳ of IRF-3 began to appear 9 hours post infection (h.p.i), and most significant activated IRF-3 appeared 23 and 26 h.p.i. Sequence analysis of NS1 of MA20 revealed that nucleotide position number 94 is mutated from T to C, and amino acid at position number 23 is changed from valine to alanine. Co-transfected with wild type NS1 made form Ⅲ and Ⅳ of IRF-3 almost disappear, but not mutant NS1. In the luciferase functional analysis, wild type NS1 can inhibit the luciferase activity of IFN-? promoter, which was induced by SV, to around 1/10. Again no inhibitory effects was observed of mutant NS1 in the luciferase assay. Conclusion The mechanism that A/HK/1/68-MA20 can activate IRF-3 is that point mutant NS1 abolished the inhibitory function of NS1.
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Objective Interferon regulatory factors 3 (IRF-3) is a key transcription factor to regulate gene expression of interferon after virus infection. This study aims to look for new spliced isoforms of IRF-3 and to investigate their structures and functions. Methods RNA extracts from human embryonic kidney 293 cells were amplified by RACE and RT-PCR. New sequences were compared with published sequences of IRF-3 and murine EST database using bioinformatics method. A new sequence, IRF-3c, was subcloned into pcDNA3.1-flag. The IRF-3c/pcDNA3.1-flag plasmid was transfected in HEK 293 cells. Whole cell extract was analysed by Western blot and then probed with monoclonal Flag antibody. Luciferase assay was carried out by co-transfection with reporter plasmid, pGL2B with interferon ? promoter, and IRF-3c cDNA expression plasmid. At 16 hours posttransfection, cells were infected with Sendai virus for 8 hours. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF-3, named IRF-3c was discovered. The new isoform is almost the same as IRF-3, except for the utilization of the 180 bp bases in intron 6 adjacent to exon 6. The first 2,3 and 4 bases are a stop codon, which may produce a protein with a truncated C-terminal stoped at amino acids 327. Western blot analysis confirmed an expected 44 kDa strong band. The new inserted bases can be found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF-3c inhibited the IFN? promoter activity to (around) 40%～50% as that of control after Sendai virus infection. Conclusions The discovery of a new isoform of IRF-3 provides a new insight into the functional regulation of IRF-3 family. It is a dominant-negative inhibitor for interferon ? promoter activity in the virus infection pathway, provides a mechanism for the fine-tuning of the virus-induced activation of the interferon response, and prevents interferon ? from its overexpression and its toxic effects. It is worthwhile to explore the role of IRF-3c in the pathogenesis of human diseases using IRF-3c’s specific sequence.