siRNA. The non-targeting (NT) siRNA was used as nega- tive control. After 48 h the medium were replaced with DMEM without FBS and cultured for another 24 h, and then siRNA transfected cells were infected with HIV-1 virus. As shown in Fig. 5a, both knockdown of p53 and knockdown of p21 by siRNA transfection were confirmed by Western blot experiment (Fig. 5a). Transfection with p21 siRNA resulted in knockdown of p21 to 13% (Fig. 5b). Transfection with p53 siRNA also led to knockdown of p21 to 18%. Transfection with p53 siRNA resulted in knockdown of p53 to 14% (Fig. 5c). To analyze the po- tential role of p53 and p21 in the regulation of genes responsible for maintaining host cell dNTPs pool size, SAMHD1, pSAMHD1(T592) and RNR2 protein levels were compared between HCT116 p53 +/+ cells trans- fected with p53 or p21 gene specific siRNA and non- targeting siRNA (Fig. 5a). It was found that the knock- down of p53 increased both RNR2 and pSAMHD1 (T- 592), and the knockdown of p21 increased RNR2 and in- creased pSAMHD1 (T-592) slightly as well. Either the knockdown of p53 or the knockdown of p21 did not change SAMHD1 at protein level significantly (Fig. 5a). The cell cycle status after siRNA treatment was also tested by Click-iT™ Plus EdU Flow Cytometry Assay. There were a minor increase in S phase in siRNA p21 treated cells (15.5%) and siRNA p53 treated cells (13.5%)
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We next sought to localize the inhibitory elements through a series of smaller substitutions. The 800 nt at the 5⬘ end of the L2 ORF were replaced by sequences from the E1 ORF to generate pPolyA Luc-EL8 (Fig. 3A). Following transfection, downstream luciferase activity was found to increase by 6.3- fold in pPolyA Luc-EL8 compared to pPolyA Luc-1500, sug- gesting that the inhibitory element was localized to this region. In contrast, when the 3⬘ half of the L2 ORF was replaced in pPolyA Luc-1500 with a comparably sized region from the E1 ORF (pPolyA Luc-LE8), no difference in downstream expres- FIG. 2. Identification of HPV-31 early polyadenylation cis elements which regulate downstream gene expression. (A) Schematic of pPolyA Luc-Control reporter construct containing a cytomegalovirus (CMV) promoter driving expression of both the Renilla (Rluc) and firefly (Fluc) luciferase genes. The genes are separated by an IRES element from encephalomyocarditis virus and contain a downstream polyadenylation signal from bovine growth hormone (bGH pA). (B) The reporter pPolyA Luc-1500 contains sequences from upstream of the E5 ORF to 1,500 nt of the late coding region. The HPV-31 sequence includes the early polyadenylation signal AAUAAA (solid vertical box) and degenerative signal UAUAUA (shaded vertical box). pPolyA Luc-P1.15 and -P2.15 contain substitutions in the AAUAAA and UAUAUA elements, respectively. pPolyA Luc-C1.15, -C2.15, and -C3.15 contain substitutions which reduce the G/U content within previously defined CstF binding sites. Plasmids were transfected into LKP-31 cells as described in Materials and Methods. Luciferase activities were determined and are illustrated graphically as the ratio of relative light units (RLU) from the downstream firefly luciferase to the upstream Renilla luciferase as a percentage of that with pPolyA Luc-Control. The ratios are presented as the standard deviations from three experiments.
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each sample was divided by the associated CAT activity to obtain a ratio of expression of the downstream to expression of the upstream gene (Table 1). The assumption was made that expression of the upstream CAT gene should be invariant and would serve as an internal standard. Relating each LUC activ- ity to its internal CAT standard should normalize the values and help detect small effects on the expression of the down- stream gene. A lower value for this LUC/CAT ratio would reflect greater attenuation, specifically the decreased expres- sion of the downstream gene relative to the upstream one. As shown in Table 1, these values were very similar from experi- ment to experiment and among the different minigenomes. To facilitate comparison, these values were then normalized within each experiment relative to that for the N/P minig- enome as 1.00 (Table 1). Within each experiment, the largest value was only 1.27- to 1.41-fold greater than the lowest one, illustrating the similarity among the different mutants. The P/M mutant exhibited the greatest LUC/CAT ratio in three of the four experiments, suggesting that the level of attenuation across this junction might be somewhat lower. The M/SH, SH/G( 2 1), and F/M2 mutants tended to have lower ratios. Since these include two of the longest intergenic regions, it is tempting to suggest that there might be a slight increase in the level of attenuation associated with increased length. However, in general, these differences were inconsistent and small, and the overall pattern among the different intergenic regions was one of close similarity. Specifically, when the results of the four experiments were averaged, the highest normalized mean LUC/CAT ratio (Table 1, right-hand column) was only 1.27- fold greater than the lowest one and all eight values were within 1 standard deviation of this mean.
. After desensitization, the receptor is internalized and targeted for degradation, redirected signalling through G-protein independent pathways (e.g. MAPK) or recycled back to the membrane . Previous studies have shown that phosphorylation site-directed mutations at the C-terminal of GPCRs severely impaired both the ability to undergo phosphorylation and to recruit arrestin [44,45]. It has also been demonstrated that con- formational changes that improve C-terminal phosphor- ylation also enhances arrestin binding and endocytosis . As depicted in Figure 1b, NPSR1-A contains more than twice as many unique phosphorylation sites as NPSR1-B. This opens up for the possibility that the -A isoform undergoes a faster turnover and hence is able to affect downstream gene expression in a more efficient way. To test this hypothesis we performed phosphoryla- tion site-directed mutagenesis on both NPSR1-A and -B, replacing all or a subset of potential phosphorylation sites with alanine. The results revealed however that the number of phosphorylation sites in the NPSR1-A and NPSR1-B C-terminal did not seem to affect the differ- ence in downstream gene regulation observed between the isoforms, even though minor effects were seen within each isoform group. The mechanisms behind isoform specific gene regulation will need further investigation.
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We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.
Global elevation of T downstream gene expression by T deletion. Because the terminator T is located just upstream of gene 11 in the middle of the T7 genome (Fig. 1A), deletion of T was expected to lead to elevated expression of T downstream genes starting from gene 11, in the middle of the genome, through gene 19.5, at the end of the genome. To test this hypothesis, reverse Northern blotting was performed using 15 DNA probes of 200 to 500 bp designed specifically for T7 genes of classes I (early), II (middle), and III (late) to semiquantitatively measure their mRNA levels in the total RNA extracts from E. coli BL21 at 10 min after infection with the wild-type or mutant phage (Fig. 3A). FIG 2 Phenotypic characterization of the T -lacking mutant phage. (A) One-step growth curve for burst size measurement of the wild-type (filled squares, solid line) and T -lacking (open circles, dash line) phages. (B) Lysis curves for estimation of lysis time from the wild-type and T -lacking mutant phage infections. (C) Adsorption rates measured for the wild-type (black column) and T -lacking (gray column) phages. Vertical error bars represent standard deviations.
Alteration of the U tract of GE2 by either increasing or decreasing its length reduced the synthesis of mRNA2, as determined both by direct labeling of RNA (Fig. 3B) and by primer extension analysis (Fig. 3C and D). For the most ex- treme U tract alterations, U14 or U2, downstream mRNA2 synthesis was reduced to very low levels compared to that from the wild-type U7 tract (Fig. 3D). Deletion of the U tract alto- gether reduced mRNA2 synthesis further still, compared to that of the U7 tract. An additional band was evident among the products of the U6-U0 mutants (Fig. 3B, lanes 6 to 9) and, as described above, this band likely resulted from infrequent ini- tiation events within the extended IGR that read through the nonfunctional GE2. While these readthrough RNAs would account for a portion of the decrease in transcription of the downstream mRNA2, they are insufficient to explain the re- maining decrease in mRNA2 production. The larger U tracts, on the other hand, would still be able to direct the termination of the IG1, -2, and -3 readthrough transcripts (5). The U8 tract directed mRNA2 synthesis to near wild-type levels, whereas the U10, U12, and U14 tracts were less efficient in directing mRNA2 production. This raised the possibility that the poly- merase has difficulty in traversing large homopolymeric se- quences, as suggested previously (5). The U tract of seven residues was previously shown to be essential for polyadenyl- ation and termination of the upstream transcript. The data presented here define a new role for the U7 tract in the effi- cient transcription of a downstream gene.
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tion of strains (17, 20, 34, 42). One lineage, described as ET1 by MLEE by Musser et al. (31) and as division I by Quentin et al. (33), is homogenous and mainly composed of serotype III strains associated with neonatal invasive disease. The MLST ST-17 presumably corresponds to this lineage. The second lineage is highly genetically diverse and includes isolates with various capsular serotypes. According to MLST, ST-19 and ST-1 also contain different capsular serotypes and have been significantly associated with the carrier state (20). We recently demonstrated that various MLST lineages were strongly asso- ciated with the presence of mobile genetic elements in the genomes of strains, probably due to horizontal gene transfers (17). This may explain why virulent subgroups of strains able to invade the CNSs of neonates more frequently possess the in- sertion sequence IS1548 in the hylB gene (encoding hyaluro- nate lyase), the group II intron GBSi1 inserted downstream from the C5a-peptidase gene scpB, and a unique cluster of tRNA genes at the 3 ⬘ end of the rRNA operons (15, 16, 34, 35).
These studies collectively provide several insights into the mechanisms of BES1- regulated gene expression. First, cis-DNA elements may help determine the function of BES1. The degenerate E-box element (CANNTG) can have multiple subtypes depending on the central two nucleotides, which are predicted to own different biochemical properties when forming hydrogen bounds with BES1 and therefore likely to affect BES1 function. In our study, BES1 shows higher affinity to the CACGTG motif and, intriguingly, this motif is very dominant in BES1 binding regions of BR-repressed genes but not in BR-activated genes. Though there are no clues to directly link the higher binding affinity to gene repression, it is possible that binding CACGTG somehow changes the structure of BES1 and poises BES1 to recruit certain protein factors and cause gene repression. This can be an interesting question for future study. Other non-E box motifs can also modulate BES1 functions through their cognate transcription factors. For example, the GAMYB binding site is found to be adjacent to BES1 binding sites in many instances. Considering the concept of enhanceosome, it is likely BES1 could form such a complex with other transcription factors to regulate gene expression cooperatively. Under such circumstances, BES1 might not always be a decisive component, which can help explain why only about one-tenth of BES1 target genes are responsive to BR stimuli.
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The reason why VACV requires so many inhibitors of NF-B activation is currently unclear but likely reflects the complex in- flammatory environment and signaling that are induced follow- ing infection in vivo. This involves significant cross talk between different innate immune-signaling pathways (45) and cell type- and host-determined specificities, all of which may play a crucial role in the outcome of infection. This is supported by the lack of redundancy of VACV NF- B inhibitors in vivo, as illustrated by the observation that in every case tested a single gene deletion caused attenuation of the virus (11, 14, 21, 24, 46–48). In vitro studies using strains of VACV with a large complement of innate immunomodulators, such as WR or Copenhagen, often fail to identify novel NF-B inhibitors during infection due to the functional redundancy observed in cell culture. Deficient vi- ruses, such as vv811, can therefore be attractive tools for the study of other aspects of host antiviral signaling, such as inhi- bition of IRF-3/7 activation. The identification of novel inhib- itors of innate immunity is an important aspect of VACV re- search and, in particular, for vaccine design. This is highlighted by recent publications reporting the enhanced immunogenic- ity of VACV strains lacking the IL-1-binding protein (encoded by B15R ), IL-18-binding protein (encoded by C12L ), and type I IFN-binding protein (encoded by B18R ) and the intracellular IRF-3/7 inhibitor C6L (52, 53), all of which are absent in vv811, thus highlighting this virus as an attractive candidate for a novel vaccine vector.
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reverse transcriptase activity, encoded by this gene, and an RNA component that serves as a template for the telomere repeat. Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells, resulting in progressive shortening of telomeres.
The food component, resveratrol, modulates lipid metabolism by activating PPARs   . Resveratrol directly interacts with PPARs and enhance their transcriptional activity . It has also been reported that soy protein de- creases blood glucose and triglyceride levels via the modulation of PPAR α path- ways  and suppresses the expression of genes related to lipid synthesis by downregulating the gene expression of SREBP1c . Although these food com- ponents and whey protein share similar properties with respect to modulating PPARs and SREBP1c, only whey protein shows potency to stimulate protein syn- thesis, differentiating it from resveratrol and soy protein.
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reduced menin gene dosage increases endocrine cell growth due to a reduction in blockage of MAPK-driven proliferation down- stream of K-RAS, while reducing K-RAS gene dosage in endocrine cells with normal menin activity increases growth by decreasing the activity of the antiproliferative RAS effector RASSF1A. This model predicts that reduced menin activity might permit acti- vators of the MAPK pathway to increase β cell proliferation. As predicted, the glucagon-like peptide 1 (GLP1) agonist exendin-4, which stimulates ERK1/2 phosphorylation in β cells (29), did not affect human β cell proliferation when added alone, but syner- gistically enhanced proliferation when combined with MI-2, an inhibitor of the interaction between menin and the histone methyl transferase MLL (ref. 30 and Figure 5, H and I).
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Several studies showed that TGF-β2 is involved in the onset and progression of several tumors, including ovarian  and pancreatic  cancer, melanoma  and malignant epidermal keratinocytes . TGF-β2 activation is a crucial step in the CD44-downstream event during tumor cell survival and proliferation . Furthermore, polymorphism in TGF-β2 promoter increased its protein expression levels, which was associated with lymph node metastasis in BC patients, thus providing evidence of the role of TGF-β2 in the process of breast tumor invasion . Epithelial-mesenchymal transition (EMT) was associated with cancer invasion and metastasis, and TGF-β signaling cascade is known to regulate EMT in the breast [80, 81]. Recent study showed that HA/CD44-moesin complex, linked with TGF-β2 receptor II and clathrin at actin microdomains, promotes activation of TGF-β signaling; this results in actin remodeling, thus causing cell-cell detachments and increased cell motility .
was evident well after the activation of LM gene, suggesting that the activated microglia might play a role in the Purkinje cell degeneration in Ngsk Prnp 0/0 mice. The elucidation of the mechanisms of the glial- cell activation in Ngsk Prnp 0/0 mice might provide in- sights into the understanding of not only the physi- ological functions of PrP C and PrPLP/Dpl but also the pathogenesis of neurodegenerative disorders, including prion diseases.
In Malaysia, Monopterus albus is commonly found in rice fields, muddy ponds and swamp areas. Channa striatus have been widely used as a source of traditional medicines. The extracts of Malaysian local Monopterus albus and Channa straitus have been reported to have different bioactive properties and these properties can be used at molecular level as alternative tool for different disorders. The comparative analysis of antibacterial, antifungal, antiproliferative and CRE induced expression of downstream luc gene activities of both the extracts were performed. The bacteriostatic and antibacterial effects of both extracts were revealed beside higher antifungal activity of Channa straitus. The extracts from Monopterus albus showed higher levels of antiproliferative activity as compare to Channastraitus. The results were found supportive towards up regulation of hrluc by Monopterus albus extracts and down regulation by Channa striatus extracts. This is the first report on comparing the bioactive properties of Monopterus albus and Channa straitus. The results from this study demands a further research on identifying the bioactive molecules involve in these actions at a molecular level.
influence its expression (Fig. 3I-P). Conversely, a -secretase inhibitor, L-685,458, which blocks cleavage and activation of Notch (Martys-Zage et al., 2000), drastically reduced mRNA levels of Nepro and Hes5, compared with the control solvent dimethylsulfoxide (DMSO), which did not affect mRNA levels (Fig. 3Q-V). Furthermore, Nepro expression was decreased by Rbpjk- specific siRNA (Fukushima et al., 2008) and the dominant-negative form of Maml1 (DN-Maml1) (Weng et al., 2003; Maillard et al., 2004) (see Fig. S5 in the supplementary material). These results indicate that Nepro is activated downstream of canonical Notch signaling.
Previous studies have shown that Notch signaling is required for normal LGE/striatal development [16,32]. Moreover, Ascl1 mutants exhibit reduced Notch signaling [14,16]. It is possible, therefore, that the phenotypes observed in the Gsx2;Ascl1 double mutants are a result of compound effects of a loss of Notch signaling together with distinct Gsx2 requirements. To address this, we examined the expression of factors in the Notch signaling pathway, Ngn2, Dll1 and Hes5, in relation to Gsx1/2 expression. In Gsx2 mutants, Ngn2 was shifted ventrally into the LGE as previously described (Figure 8F) [1-3], although it appeared to be directly abutting the ventrally shifted Gsx1/2 staining (Figure 8B). Indeed, both Dll1 (Figure 8J) and Hes5 (Figure 8N) were continuously expressed throughout the Gsx2 mutant LGE. In the Ascl1 mutants, Gsx1/2 staining was present up to the normal pallio-subpallial boundary (Figures 3A and 8C) and Ngn2 staining abutted it at its normal ventral position (compare Figure 8E and 8G). This theoretically leaves no proneural gene expression in the LGE and, in fact, both Dll1 (Figure Removal of Ascl1 on the Gsx2 mutant background exacerbates the Gsx2 mutant phenotype in the striatum
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existence of a bimodal pattern in mRNA distribution in the expo- nential phase, which is not observed in other phases; this is in agreement with the observed mRNA distribution of the lac pro- moter (23). To determine whether the bimodal behavior is regu- lated by the URS/DRS of the promoter, we activated the URS and DRS of the lac variant independently (see Fig. S1 and Table 1 at http://www.cs.tut.fi/~kandhave/supplementary/Supplementary %20Material.pdf). Figure 1c shows a unimodal distribution of mRNA in all phases, during which the independent activation of the URS is capable of producing low numbers of mRNA (1 to 2 molecules), apparently in a single-grade mode. Moreover, the DRS produced a broad range of mRNAs (1 to 20 molecules); we also observed the existence of a single-grade mode alone (Fig. 1c). We also noticed that in a population, the activated URS and DRS tend to produce low and high numbers of mRNA, respectively (Fig. 1c). It is possible that the URS has a stronger RNAP binding site and a weaker promoter than the DRS, which is in close agree- ment with previous observations (19, 20, 24). Notably, when cells receive inducers to activate both the URS and the DRS, the pro- moter regulates the production of a small number (1 to 2 mole- cules) and a large number ( ⬎ 2 molecules) of mRNAs at low and high frequencies, respectively (Fig. 1b). Activation appears to oc- cur in a binary mode, confirming the existence of two distinct populations (Fig. 1b). Thus, we conclude that the bimodal mRNA distribution results from the coordinated regulation by URS/DRS gene activation. We suggest that there are multiple stochastic events in promoter transition that trigger phenotypic switching (1); this may be due to the competitive binding of RNAP at these sites (24). Moreover, we see the same general features of mRNA distribution in the lac variant as in endogenous lac, which further supports our conclusion regarding the bimodal behavior due to the complexity of promoter transition (Fig. 1a and b). We also observed that bias in the partitioning of RNAs does not lead to phenotypic switching (Fig. 1e), despite the change in the fre- quency of mRNA production (Fig. 1f), which in turn triggers the bimodality.
TCR signaling pathways cooperate to activate the inducible transcription factors NF-kB, NFAT, and AP-1. In this study, using the calcium ionophore ionomycin and/or PMA on Jurkat T cells, we show that the gene expression program associated with activation of TCR signaling is closely related to specific chromatin landscapes. We find that calcium and kinase signaling cooperate to induce chromatin remodeling at ∼ 2100 chromatin regions, which demonstrate enriched binding motifs for inducible factors and correlate with target gene expression. We found that these regions typically function as inducible enhancers. Many of these elements contain composite NFAT/AP-1 sites, which typically support cooperative binding, thus further reinforcing the need for cooperation between calcium and kinase signaling in the activation of genes in T cells. In contrast, treatment with PMA or ionomycin alone induces chromatin remodeling at far fewer regions ( ∼ 600 and ∼ 350, respectively), which mostly represent a subset of those induced by costimulation. This suggests that the integration of TCR signaling largely occurs at the level of chromatin, which we propose plays a crucial role in regulating T cell activation. The Journal of Immunology, 2017, 199: 2652–2667.
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