Chloramphenicol Acetyltransferase Expression Research Articles

The effect of dermatophytes on cytokine production by human keratinocytes

Dermatophytosis (tinea) is a common disease in superficial mycoses and is generally confined to the stratum corneum in the epidermis and cutaneous appendages. The mechanisms by which dermatophytes cause dermatophytosis, however, are poorly understood. In this study, we evaluated the effect of Trichophyton mentagrophytes, T. tonsurans and T. rubrum on cytokine production by normal human epidermal keratinocytes (NHEKs). After 3-24 h of co-culture of NHEKs with each of the dermatophytes, cytokines in the supernatant were measured by enzyme-linked immunosorbent assay. Promoter activity of IL-8 was measured by chloramphenicol acetyl transferase (CAT) assay. IL-8 and GRO-alpha levels were higher in supernatants co-cultured with T. mentagrophytes isolates from animal than in those with T. mentagrophytes isolates from human, and with T. tonsurans and T. rubrum isolates. CAT expression for IL-8 promoter activity was higher in cell lysates stimulated with T. mentagrophytes isolates from animal than in those with T. mentagrophytes isolates from human, and with T. tonsurans and T. rubrum isolates. These findings suggest that dermatophytes directly induce production of cytokines at the transcriptional level by human keratinocytes, and that there are differences in their ability to induce cytokine production between the dermatophytes.

Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element

Chimeric oligo-2′- O-methylribonucleotides containing centrally located patches of contiguous 2′-deoxyribonucleotides and terminating in a nuclease resistant 3′-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3′-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA ( K D approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24 h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.

Increasing transient expression of CAT gene in Porphyra haitanensis by Matrix attachment regions and 18S rDNA targeted homologous recombination

To test whether matrix attachment regions (MARs) and 18S rDNA can in£uence CAT gene transient expression positively in the red algae Porphyra haitanensis, a targeting vector pHR-CAT containing a portion of the 18S rDNA from P. haitanensis ,p MAR1-HR-CAT containing one MAR from silkworm and a portion of the 18S rDNA from P. haitanensis and pMAR2-HRCAT containing two MARs from silkworm and a portion of the 18S rDNA from P. haitanensis were constructed. With the electroporation method, the vectors were transferred into the protoplasts from the thalli of P. haitanensis. The results showed that the expression of chloramphenicol acetyl transferase (CAT) protein in transformed cells reached a maximum at 96 h after transformation. It was increased markedly with the pMAR2-HR-CAT compared with the pHR-CAT or the pCAT @ 3-control vector (Po0.01), and it was increased inconspicuously with pHR-CAT compared with the pCAT @ 3-control vector (P40.05). It is suggested that MAR from silkworm could enhance the transient expression of foreign genes in P. haitanensis.

Optimizing Aerosol Gene Delivery and Expression in the Ovine Lung

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.

Cloning and Characterization of Promoter-Active DNA Sequences from Streptococcus equisimilis

Genomic fragments of Streptococcus equisimilis exhibiting potent promoter activity in Escherichia coli were isolated by transcriptional fusion to chloramphenicol acetyl transferase (CAT) structural gene. Random S. equanimities DNA, cloned in E. coli, exhibited a higher frequency of strong promoter activity than did similarly cloned E. coli fragments. The determination of the relative promoter strength of randomly selected clones with CAT assay demonstrated the dominance of sequences acting as a strong promoter in E. coli. Removal of downstream terminator from the strong promoter containing plasmid resulted in a twofold to threefold increase in CAT expression in some cases. Structural characteristics of promoter sequences of some representative streptococcal genes clearly indicate that the streptococcal promoter regions are rich in secondary structures and might be one of the factors of their instability in E. coli.

The transcriptional activity of HERV-I LTR is negatively regulated by its cis-elements and wild type p53 tumor suppressor protein

Human endogenous retroviruses (HERVs), abundantly inter-dispersed in the genome, carry long terminal repeats (LTRs) that may potentially retro-transpose to new genomic sites and deregulate the neighboring cellular genes. However, normally HERVs are either structurally defective or inactive due possibly to stringent negative control mechanisms. To study the possible negative regulation of HERV, we isolated the LTR of RTVL-Ia and constructed site-specific mutations for analysis of the promoter and enhancer functions by using chloramphenicol acetyl transferase (CAT) reporter assay. Our results showed that in most transfected human cells the LTR-mediated CAT expression was negligible unless a sequence segment at the AGTAAA polyadenylation site was deleted. In addition, we have found that the wild type p53 may inhibit whereas a p53 mutant (V143A) stimulate the transcriptional activity of HERV-I LTR. Our results imply that HERV-I LTR, while under negative control by its LTR cis-elements and by wild type p53, may become active upon p53 mutation.

PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA

BackgroundThe HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNA through interaction with the Rev response element (RRE) by means of an arginine rich motif that is similar to the one found in Tat. Since Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its arginine rich motif, we investigated whether the Rev protein could act as a substrate for this enzyme.ResultsHere, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner.ConclusionThese findings indicate that PRMT6 severely impairs the function of HIV-1 Rev.

The Role of pp38 in Regulation of Marek’s Disease Virus Bi-directional Promoter between pp38 and 1.8-kb mRNA

Marek's disease virus (MDV) contains a bi-directional promoters located between pp38 gene and 1.8-kb mRNA in the long inverted repeat region of the viral genome. The involvement of pp38 gene in up-regulating the activity of these promoters was analyzed by transient expression of chloramphenicol acetyltransferase (CAT) reporter gene. Two CAT reporter plasmids, pP(pp38)-CAT and pP(1.8-kb)-CAT, were constructed to express CAT under the control of the bi-directional promoter in both orientations. These plasmids were transfected into chicken embryonic fibroblast (CEF), infected with rMd5 and pp38 deleted rMd5 (rMd5/Deltapp38), respectively. No CAT activity was detected in uninfected CEF as expected. CAT activities in rMd5/Deltapp38 virus infected CEF (rMd5/Deltapp38-CEF) were 3.5-fold lower using pP(pp38)-CAT and 12-fold lower using pP(1.8-kb)-CAT than those of the parental rMd5 infected CEF (rMd5-CEF). The significantly lower promoter activity in the pp38 deletion virus suggests that pp38 can regulate the activity of the bi-directional promoters, especially in the direction of 1.8-kb mRNA family. Co-transfection of pp38-expressing plasmid (pcDNA-pp38) into rMd5/Deltapp38-CEF significantly increased the activity of the bi-directional promoters using either pP(pp38)-CAT or pP(1.8-kb)-CAT. DNA mobility shift assay showed a binding of the 73-bp sequence of the bi-directional promoter with rMd5-CEF but not with rMd5/Deltapp38-CEF or uninfected CEF lysates. However, rMd5/Deltapp38-CEF lysates could bind the same 73-bp promoter sequence when co-transfected with pp38-expressing plasmid (pcDNA-pp38). All these data taken together suggest pp38 plays an important role in regulating the transcriptional activity of the bi-directional promoter.

Interactions between Areas I and II Direct pdx-1 Expression Specifically to Islet Cell Types of the Mature and Developing Pancreas

PDX-1 regulates transcription of genes involved in islet beta cell function and pancreas development. Islet-specific expression is controlled by 5'-flanking sequences from base pair (bp) -2917 to -1918 in transgenic experiments, which encompasses both conserved (i.e. Area I (bp -2761/-2457), Area II (bp -2153/-1923)) and non-conserved pdx-1 sequences. However, only an Area II-driven transgene is independently active in vivo, albeit in only a fraction of islet PDX-1-producing cells. Our objective was to identify the sequences within the -2917/-1918-bp region that act in conjunction with Area II to allow comprehensive expression in islet PDX-1(+) cells. In cell line-based transfection assays, only Area I effectively potentiated Area II activity. Both Area I and Area II functioned in an orientation-independent manner, whereas synergistic, enhancer-like activation was uniquely found with duplicated Area II. Chimeras of Area II and the generally active SV40 enhancer or the beta cell-specific insulin enhancer suggested that islet cell-enriched activators were necessary for Area I activation, because Area II-mediated stimulation was reduced by the SV40 enhancer and activated by the insulin enhancer. Several conserved sites within Area I were important in Area I/Area II activation, with binding at bp -2614/-2609 specifically controlled by Nkx2.2, an insulin gene regulator that is required for terminal beta cell differentiation. The ability of Area I to modulate Area II activation was also observed in vivo, as an Area I/Area II-driven transgene recapitulated the endogenous pdx-1 expression pattern in developing and adult islet cells. These results suggest that Area II is a central pdx-1 control region, whose islet cell activity is uniquely modified by Area I regulatory factors.

The influence of cold shock proteins on transcription and translation studied in cell‐free model systems

Cold shock proteins (CSPs) form a family of highly conserved bacterial proteins capable of single-stranded nucleic acid binding. They are suggested to act as RNA chaperones during cold shock inhibiting the formation of RNA secondary structures, which are unfavourable for transcription and translation. To test this commonly accepted theory, isolated CSPs from a mesophilic, thermophilic and a hyperthermophilic bacterium (Bacillus subtilis, Bacillus caldolyticus and Thermotoga maritima) were studied in an Escherichia coli based cell free expression system on their capability of enhancing protein expression by reduction of mRNA secondary structures. The E. coli based expression of chloramphenicol acetyltransferase and of H-Ras served as model systems. We observed a concentration-dependent suppression of transcription and translation by the different CSPs which makes the considered addition of CSPs for enhancing the protein expression in in vitro translation systems obsolete. Protein expression was completely inhibited at CSP concentrations present under cold shock conditions. The CSP concentrations necessary for 50% inhibition were lowest (140 microm) for the protein of the hyperthermophilic and increased when the thermophilic (215 microm) or even the mesophilic protein (451 microm) was used. Isolated in vitro transcription under the influence of CSPs showed that the transcriptory effect is independent from the rest of the cell. It could be shown in a control experiment that the inhibition of protein expression can be removed by addition of hepta-2'-desoxy-thymidylate (dT7); a heptanucleotide that competitively binds to CSP. The data are in line with a hypothesis that CSPs act on bulk protein expression not as RNA chaperones but inhibit their transcription and translation by rather unspecific nucleic acid binding.

Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation

Alpha interferons (alpha-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of alpha-IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of alpha-IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by alpha-IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other alpha-IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.

Characterization of an upstream regulatory element of adenovirus L1 poly (A) site

The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1–L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532–541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977–5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3′ cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682–4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3′ end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207–2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state mRNA expression from the MLTU L3 poly (A) site or the murine β-globin poly (A) site. In the tandem L13 mini-gene system, the L1URE is revealed to influence steady state mRNA stability, and subdomains within L1URE influence both gene expression and the steady state ratio of transcripts processed at proximal versus distal poly (A) sites. Transcript and gene repression mediated by the L1URE may provide a general strategy employed by adenovirus to block premature expression of late stage MLTU transcripts.

Genetically engineered mouse melanoma and mouse Lewis lung carcinoma models

Melanoma B16-F10 cells and Lewis lung carcinoma LL/2 cells were engineered with a bacterial gene -- chloramphenicol acetyl transferase (CAT) -- by establishing stable transductants. Expression of CAT in both cell types did not alter the ability of these cells to grow into tumors when injected subcutaneously into mice. In addition, the measurement of CAT levels in the lung using a simple ELISA assay revealed a close correlation with direct counting of metastatic nodules. Thus, the CAT-expressing cells will likely have wide ranging applications to quantify tumor metastasis especially in situations where visual counting is difficult. The availability of genetically labeled mouse B16-F10 melanoma and Lewis lung carcinoma cell lines will facilitate future studies of the mechanism and progression of cancer and the discovery of new therapies.

Biological impact of the COOH-terminal 40 amino acid deletions of hepatitis B virus X protein in hepatocellular carcinoma cells

To investigate the biological impact of the wild type hepatitis B virus X protein (HBx) and HBx3'-40, an engineered deletion mutant of HBx lacking the last 40 C-terminal amino acids and a transcriptional transactivator on hepatoma cells. Human hepatocellular cells of the lines Huh7 and SMMC-7721 were transfected with HBx3'-40 or HBx constructs. The integration of the plasmid DNA and the expression of HBx3'-40 and HBx were confirmed by Neo gene PCR and Western blotting respectively. The growth curves of different cells were obtained by MTT method. Plate clone formation test to calculate the clone formation rate. Flow cytometry (FCM), and Chloramphenicol acetyl transferase (CAT)-ELISA. Male BALB/c mice were divided into 3 groups to be inoculated subcutaneously with plasmids pcDNA3HBx, pcDNA3HBx3'-40, and blank plasmas pcDNA3 as controls. The mice were killed 15 and 30 days after and the tumors were taken out to undergo measurement and immunohistochemistry. Growth curve showed that after transfection the HBx3'-40 group cells grew faster than HBx and pcDNA3 transfected group cells. FCM analysis showed that HBx3'-40 transfection enhanced the progression of G(1) --> S phase in Huh7 cell cycle and HBx did not show such impact. The clone formation rates of pcDNA3HBx3'-40 group was 12.2% +/- 1.4%, significantly higher than those of the pcDNA3HBx, blank vector, and control groups (6.3% +/- 0.7%, 4.1% +/- 0.9%, and 6.1% +/- 1.1% respectively, all P < 0.05) in the Huh7 cells. The situation was the similar in the SMMC-7721 cells: 83% +/- 6% vs.49% +/- 8%, 27% +/- 3%, and 51% +/- 5% respectively (all P < 0.05). The apoptotic rates of SMMC-7721 cells under no serum condition transfected with HBx and HBx3'-40, especially the former, were 12.57% and 9.15%, significantly higher than that of the pcDNA3HBx group. The CAT expression of the pcDNA3HBx-transfected SMMC-7721 cells was 2.913 and 3.652 times those of the pcDNA3HBx3'-40 group and pcDNA3 group. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected Huh7 cells were 1.2 g +/- 0.17 g, 0.55 g +/- 0.12 g, and 0.48 g +/- 0.15 g respectively. The tumor weights of the mice inoculated with pcDNA3HBx3'-40, pcDNA3HBx, and blank vector-transfected SMMC-7721 cells showed similar features. HBx3'-40 protein significantly promote the proliferation of hepatocellular carcinoma cells and is involved in cell apoptosis and invasion, thus supporting the hypothesis that HBx mutants play a key role in hepatocarcinogenesis by modifying the biological functions of HBx. Besides, C-terminal mutants of HBx makes p53 tumor suppressor gene lose its cancerostatic function.

Human interleukin-1α gene expression is regulated by Sp1 and a transcriptional repressor

The regulation of the human IL-1α gene was studied using a series of 5′ deletion promoter chloramphenicol acetyltransferase (CAT) reporter constructs. The IL-1α promoter from −967 to +64 produced no significant expression of CAT. Progressive 5′ deletion indicated the presence of a repressor binding site between −477 and −305 bp as deletion in this region resulted in CAT expression. Electrophoretic mobility shift assay (EMSA) analysis confirmed that protein(s) bound to this region and DNaseI footprinting localized the binding site to between −448 and −435. Deletion of the IL-1α promoter to −42 resulted in reduced CAT expression suggesting the presence of a positive regulatory element in this region. EMSA experiments using IL-1α promoter DNA from −163 to +64 demonstrated protein binding to this region and DNaseI footprinting demonstrated protection between −59 and −40. Transcriptional activity of the IL-1α promoter was also tested using an in vitro transcription assay. Reactions using −163, −100 and −52 promoter templates all produced a correctly sized transcript but deletion to −42 resulted in no transcript production. Analysis of the promoter indicated that a potential Sp1 binding site existed in the region from −52 to −45. An EMSA using an anti-Sp1 antibody indicated that Sp1 specifically bound to the −52 to +64 region.

Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics

We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Baicalin induces NAD(P)H:quinone reductase through the transactivation of AP-1 and NF-κB in Hepa 1c1c7 cells

Baicalin (5,6,7-trihydroxyflavone-7-O-D-glucuronic acid, BA) is a flavone isolated from Scutellariae radix. In our previous report BA was a major active principle of NAD(P)H:quinone reductase (QR) induction mediated by Scutellariae radix extract and the induction was related to the transcriptional activation of the QR gene in Hepa 1c1c7 cells. The primary aim of the present study was to determine the molecular mechanism of QR gene expression by baicalin. The antioxidant or electrophile response element (ARE/EpRE) found at the 5'-flanking region of phase II genes may play an important role in mediating their induction by xenobiotics, including chemopreventive agents. In accordance, to study the molecular mechanisms of QR gene expression by BA, electrophoretic mobility shift assay (EMSA), using nuclear extracts of treated and untreated cells against ARE, activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) binding sites, showed that BA increased the binding levels of the parameters in a dose-dependent manner. Further, Hepa 1c1c7 cells were transiently transfected with a plasmid containing three copies of the AP-1- or NF-kappaB-binding site linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Using the CAT reporter gene assay, a dose-dependent transactivation of AP-1- or NF-kappaB-mediated CAT expression was observed with the treatment of BA. These results clearly indicate that BA induces the QR gene expression and activity by transactivation of AP-1 and NF-kappaB, and thus BA may be considered as a potential cancer chemopreventive agent with the induction of phase II detoxification enzyme.

The activity of the feline thyroglobulin promoter is compromised by flanking adenoviral sequence

The use of human adenovirus (Ad) vectors for transcriptionally targeted thyroid gene therapy was investigated, aiming to develop a novel therapy for feline hyperthyroidism. Ad5 (El, E3 deleted) vectors were constructed in which the feline thyroglobulin promoter controls expression of the reporter gene chloramphenicol acetyl transferase (CAT). In FRTL-5 cells, CAT expression from adenoviral constructs harbouring the expression cassette was much reduced compared to controls transfected with the same cassette in a plasmid backbone, despite higher transduction efficiencies when viral vectors were used. Transfections with the “shuttle” plasmids utilised to create the adenoviral vectors also resulted in reduced expression compared to controls. In both viruses and shuttle plasmids, consistently lower expression was noted when the cassette was in the left to right orientation than the right to left. These results suggest cis acting elements in the flanking adenoviral sequences may compromise the activity of the feline thyroglobulin promoter and thus make Ad5 an unsuitable vector for transcriptionally targeted gene therapy in feline hyperthyroidism.

Corticotropin-releasing hormone stimulates NF-kappaB in human epidermal keratinocytes.

Corticotropin-releasing hormone (CRH) has been shown to inhibit proliferation and modulate expression of inflammation markers in the epidermal cells. In the present study we report that CRH also stimulates nuclear factor-kappa B (NF-kappaB) activity. Incubation with CRH of human keratinocytes derived from primary cultures resulted in increased binding of DNA by NF-kappaB. CRH induced translocation of NF-kappaB subunit p65 from the cytoplasm to the nucleus and induced expression of kappaB-driven chloramphenicol acetyltransferase (CAT) reporter gene. NF-kappaB translocation was accompanied by degradation of the inhibitor of NF-kappaB alpha (IkappaB-alpha). Specificity of the CRH effect was demonstrated by the use of CRH-R antagonists antalarmin and alpha-helical CRH [9-41]. CRH-dependent stimulation of NF-kappaB activity is consistent with accumulated data about role of this neuropeptide in the regulation of local epidermal homeostasis.