High IFN-α expression is associated with the induction of experimental autoimmune uveitis (EAU) in Fischer 344 rat

Th1-response plays a crucial role in determining pathogenesis of organ-specific autoimmune diseases. It is believed that both IL-12 and INF-alpha are initiators to regulate Th1-response. In our experimental autoimmune uveitis (EAU) model, both Lewis and Fischer 344 rats share the same MHC class II molecules, while Lewis rat is EAU susceptible and Fischer 344 rat is EAU resistant. However, under the same condition of immunization, if pertussis toxin (PTX) was injected intraperitoneally as an additional adjuvant, Fischer 344 rat can develop EAU. In this study we investigate which mechanisms are involved in the induction of EAU in CFA+R16+PTX-treated (CRP-treated) Fischer 344 rats. In vivo and in vitro data demonstrated that Th1-cytokine, IFN-gamma mRNA expression was significantly increased in disease target tissue-eyes and in draining lymph node cells of CRP-treated Fischer 344 rat. When IL-12 and IFN-alpha mRNA expression were compared in the experimental groups, only IFN-alpha mRNA expression was associated with EAU development. To distinguish the sources of IFN-alpha producing cells, it was observed that IFN-alpha expression was mainly produced by macrophages. It was further confirmed that normal macrophage from Fischer 344 rat was able to produce significant IFN-alpha in the presence of PTX. The data strongly suggested that IFN-alpha might be involved in initiating Th1-cell differentiation and in turn contribute to the induction of EAU. High IFN-alpha expression induced by PTX may represent a novel pathway to initiate Th1 response in Fischer 344 rat.

Association of <i>NOS3</i>and <i>HIF</i>1<i>α</i>gene polymorphisms with the susceptibility of broiler chickens to develop hypoxic pulmonary hypertension

A genetic association between single nucleotide polymorphisms (SNPs) and pulmonary hypertension syndrome (PHS) was established in a commercial population of broiler chickens. The associated SNPs were found in the NOS3 and HIF1α genes (LOD > 6;p NOS3 gene interfere with its trans-activation and transcriptional activation activities under natural hypobaric hypoxia conditions and are located in a consensus sequence that is called the hypoxia response element (HRE). SNPs located in the HIF1α gene could act as alternative cryptic splicing sites in intron six, which may stimulate non-sense mediated early decay (NMD) of the primary transcript. A fragment of intron 3 of the EDN1 gene was also evaluated, but the polymorphisms found were not associated with PHS (lod 0.001). However, further studies on the regulatory transcription sequences of EDN1 are recommended. The findings of this study indicate that intronic sequences should be included when searching for polymorphisms that produce physiological changes. Introns have transcriptional regulatory sequences or post-transcriptional control signals, which are known as cis- and trans-activation regulatory elements and are able to alter the physiological processes of hypoxia adaptation when modified. Based on these findings, it can be concluded that the inheritance pattern of PHS is autosomal overdominant and has deleterious effects that are characterized by higher penetrance in heterozygous than in homozygous animals, which prevent broiler chickens from being able to adapt to high altitudes.

The 5’-Untranslated Region of the C9orf72 mRNA Exhibits a Phylogenetic Alignment to the Cis-Aconitase Iron-Responsive Element;Novel Therapies for Amytrophic Lateral Sclerosis

The hexanucleotide repeat mutation in the intron-1 of the chromosome 9 open reading frame (C9orf72) is a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Altered RNA folding plays a role in ALS pathogenesis in two ways: non-ATG translation of the repeat can lead to aggregates of the known C9orf72 specific dipeptide polymer, whereas the repeat also can form neurotoxic RNA inclusions that dose-responsively kill motor neurons. We report the presence of a homology in the 5’untranslated region (UTR) of the messenger RNA encoding C9orf72 with the iron responsive elements (IRE) that control expression of iron-associated transcripts and predict that this RNA structure may iron-dependently regulate C9orf72 translation. We previously report altered serum ferritin levels track with severity of ALS in patients. Here, we conduct bioinformatics analyses to determine the secondary structure of the 5’UTR in C9orf72 mRNA and find it aligned with IREs in the human mitochondrial cis-aconitase and L and H-ferritin transcripts. Comparison of the role of RNA repeats in Friedriech’s ataxia and fragile X mental retardation suggests the utility of RNA based therapies for treatment of ALS. Antisense oligonucleotides (ASO) have been reported to therapeutically target these GGGGCC repeats. At the same time, because the function of C9orf72 is unknown, knockdown strategies carry some risk of inducing or compounding haploinsufficiency. We propose, for consideration, an approach that may enhance its therapeutic dynamic range by increasing the 5’UTR driven translation of C9orf72 protein to compensate for any potential ALS-specific or ASO-induced haploinsufficieny.

Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44MAPK, p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44MAPK, p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44MAPK and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between p42/44MAPK and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Rat/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44MAPK, c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

Borf-1 protein identified as a transcriptional trans-activator of bovine foamy virus

Bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses,contains two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag,po/and env. Borf-1 protein, encoded by BFV ORF-1, is identified as a transcriptional transactivator, which augments gene expression directed by the viral long terminal repeat (LTR).Further investigations in transient expression assays reveal that the Borf-1 responsive elements are located in the U3 domain of the LTR, upstream from position -140 ( + 1 represents the transcription initiation site), and the BFV RU5 region has an inhibitory effect in LTR-directed gene expression.

Review of current progress in the structure and function of Smad proteins

PURPOSE: To review the recent developments in the structure and function of Smad proteins. DATA SOURCES: Both Chinese- and English-language literatures were searched using MEDLINE/CD-ROM (1997 - 2000) and the Index of Chinese-Language Literature (1997 - 2000). STUDY SELECTION: Data from published articles about TGF-beta signal transduction in recent domestic and foreign literature were selected. DATA EXTRACTION: Data were mainly extracted from 22 articles which are listed in the reference section of this review. RESULTS: Smad proteins mediate signal transduction induced by the TGF-beta superfamily. Based on their structural and functional properties, Smad proteins are divided into three groups. The first group, receptor-regulated Smads (R-Smads), are phosphorylated by activated type I receptors and form heteromeric complexes with the second group of Smads, common mediator Smads (Co-Smads). These Smad complexes translocate into the nucleus to influence gene transcription. Inhibitory Smads (I-Smads) are the third group and these antagonize the activity of R-Smads. In the nucleus, Smads can directly contact Smad-binding elements (SBE) in target gene promoters. Through interaction with different transcription factors, transcriptional co-activators or co-repressors, Smads elicit different effects in various cell types. The aberrance of Smad proteins has been noted in several human disorders such as fibrosis, hypertrophic scarring and cancer. CONCLUSION: The structure of Smads determines their function as transcriptional factors which translocate signals from the cell surface to the nucleus where Smads regulate TGF-beta superfamily-dependent gene expression.

Expression of interleukin-12 and its signaling molecules in peripheral blood mononuclear cells in systemic lupus erythematosus patients

Objective To determine the in vitro expression of interleukin-12 (IL-12) and its effect on signal transducers and activators of transcription (STAT) signaling molecules in peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells in 39 patients with definite systemic lupus erythematosus and 11 healthy volunteers were collected. Expression of IL-12 P40mRNA in PBMCs was determined with reverse transcription-polymerase chain reaction (RT-PCR). Quantity of IL-12 protein supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The levels of phosphorylated STAT3 and STAT4 signaling molecules in PBMCs were detected by immunoblot. Results Levels of IL-12 protein and mRNA expression in patients with active or inactive SLE were significantly higher than those in controls. Phytohemagglutinin (PHA) may promote the expression of IL-12. IL-12 alone induced the phosphorylation of STAT3 and STAT4 in PBMCs from patients with SLE, especially in active SLE. However it had no obvious effect on normal PBMCs. Phosphorylated STAT3 and STAT4 might be observed in normal PBMCs treated with IL-12 plus PHA.Conclusion IL-12 is produced aberrantly in patients with SLE. IL-12 might exert its biological role in SLE via the aberrantly phosphorylated STAT3 and STAT4 signaling molecules.

Cloning and polymorphism analysis of IL-4 proximal promoter in asthmatic children

OBJECTIVE: To clone and study the polymorphism within interleukin-4 (IL-4) proximal promoter of asthmatic children. METHODS: The IL-4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial histories as templates. The selected PCR segments were cloned into recombinant plasmids pIL-4-Jx2. The PCR inserts were sequenced by dideoxy chain termination method. RESULTS: Seven aberrant bands were found in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL-4 regulatory element. A C to A transversion located at -229 position was just within the positive regulatory element-I (PRE-I) in one patient. A C to T transition adjacent to the negative regulatory element-II (NRE-II) and an extra G adjacent to TATA box were found in two patients. A five base nucleotide deletion was found near signal transducers and activators of transcription-6 responsive element (STAT-6 RE) in one patient. CONCLUSION: There were polymorphisms within the IL-4 proximal promoter of allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL-4 gene and asthma.

Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression

Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering.Along the way,combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits.In this report,we present an efficient evolutionary approach to build a range of regulatory control elements.The reported method allows for rapid construction of promoter,5'UTR,terminator and trans-activating RNA libraries.Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter.This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix.Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics.Specifically,both the promoter library and 50UTR library exhibits gene expression dynamics spanning across three order of magnitude.The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern.The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements.These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.

On the structure of AP-4 responsive element in the LTR of Jembrana disease virus

Previous studies with deletion and sequence analysis of JDV LTR showed that there is a putative AP-4 responsive element in LTR. By antisense transient assay and gel shifting assay, we for the first time demonstrated that AP-4 modulated JDV gene expression by binding DNA di-rectly to bovine cells. The results, derived from site-directed mutagenesis experiments, suggest that the six base pairs of AP-4 binding site (CAGCTG) have different effects on JDV gene expression. When the first two base pairs changed to GC, JDV gene expression is severely decreased.