Down-regulation of peroxisome proliferator-activated receptor-gamma gene expression by sphingomyelins

We recently demonstrated that the sphingomyelin (SM) content of adipocyte membranes was negatively correlated with the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) in the subcutaneous adipose tissue of obese women with variable degrees of insulin resistance. We have now investigated whether SM really does have an impact on the expression of PPARgamma in 3T3-F442A adipocytes. Adding SM to the culture medium for 24 h caused a significant increase in SM content of adipocyte membranes and an acyl chain length-dependent decrease in the levels of PPARgamma mRNA and protein. The longer the acyl chain of the fatty acid of SM, the greater was the decrease in PPARgamma. These data suggest that the nature of the fatty acid is important in the regulation of PPARgamma by the SM pathway.     

Visualization of membrane rafts using a perylene monoimide derivative and fluorescence lifetime imaging

A new membrane probe, based on the perylene imide chromophore, with excellent photophysical properties (high absorption coefficient, quantum yield (QY) approximately 1, high photostability) and excited in the visible domain is proposed for the study of membrane rafts. Visualization of separation between the liquid-ordered (Lo) and the liquid-disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the membrane probe in the two phases. Rafts on micrometer-scale in cell membranes due to cellular activation can also be observed by this method. The decay time of the dye in the Lo phase is higher than in organic solvents where its QY is 1. This allows proposing a (possible general) mechanism for the decay time increase in the Lo phase, based on the local field effects of the surrounding molecules. For other fluorophores with QY<1, the suggested mechanism could also contribute, in addition to effects reducing the nonradiative decay pathways, to an increase of the fluorescence decay time in the Lo phase.     

Down-regulation of Notch target gene expression by Suppressor of deltex

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.     

Down-regulation of hepatic lipase gene expression and activity by fenofibrate.

The influence of the hypolipidemic drug, fenofibrate, on hepatic lipase (HL) gene expression and activity was investigated in the rat. Fenofibrate treatment provoked a dose-dependent decrease in HL mRNA levels. At a dose of 0.5% (w/w), HL mRNA levels were reduced to nearly 50% the levels in untreated controls. This decrease was parallelled by a comparable reduction in liver HL activity. The decrease in HL mRNA levels was already observed after 1 day of fenofibrate treatment. Whole liver perfusion experiments showed that the heparin-releasable HL activity in fenofibrate-treated livers dropped to 10% the activity in control livers. In conclusion, treatment with fenofibrate decreases HL gene expression, leading to a lowered activity of endothelium bound HL in fenofibrate-treated livers.     

Exercise adaptation attenuates VEGF gene expression in human skeletal muscle

Angiogenesis is a component of the multifactoral adaptation to exercise training, and vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation. However, there is limited evidence supporting the role of VEGF in the exercise training response. Thus we studied mRNA levels of VEGF, using quantitative Northern analysis, in untrained and trained human skeletal muscle at rest and after a single bout of exercise. Single leg knee-extension provided the acute exercise stimulus and the training modality. Four biopsies were collected from the vastus lateralis muscle at rest in the untrained and trained conditions before and after exercise. Training resulted in a 35% increase in muscle oxygen consumption and an 18% increase in number of capillaries per muscle fiber. At rest, VEGF/18S mRNA levels were similar before (0.38 +/- 0.04) and after (1.2 +/- 0.4) training. When muscle was untrained, acute exercise greatly elevated VEGF/18S mRNA levels (16.9 +/- 6.7). The VEGF/18S mRNA response to acute exercise in the trained state was markedly attenuated (5.4 +/- 1.3). These data support the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis and appears to be subject to a negative feedback mechanism as exercise adaptations occur.     

抗人血管内皮生长因子单链抗体基因的构建、表达及活性鉴定

鼠单克隆抗体E11能与人血管内皮生长因子(VEGF)特异结合,已用于临床检测恶性肿瘤细胞VEGF的表达,并初步证明其体内抑瘤活性。为便于大规模生产,特进行基因工程改造,构建小分子的单链抗体(scFv)。首先通过逆转录及多聚酶链式反应(PCR),分离并克隆E11的可变区基因。经测序表明,E11轻链可变区(VL)基因全长333bp,编码111个氨基酸,归属小鼠轻链可变区基因第Ⅲ亚组。重链可变区VH基因全长369bp,编码123个氨基酸,归属小鼠重链可变区基因Ⅱ(A)亚组。然后用一编码亲水性多肽接头的DNA片断将E11单抗轻、重链可变区基因连接,构建表达质粒pET-15YV,在大肠杆菌BL21(DE3)中进行表达。表达产物(包含体)经变性及复性后,用免疫组化法检测该单链抗体结合抗原(颊癌)能力。对颊癌组织检测的结果表明,基因工程抗体scFv与亲代抗体一样,具有较高的组织特异性。本研究获得的抗人VEGF单链抗体具有潜在的临床价值,为肿瘤放射免疫显像及以血管为靶标的抗血管生成治疗奠定了基础。     

Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC₅₀ values of HPDIs were in the low micromolar (0.5–5 μM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.     

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

Down-regulation of GhADF1 gene expression affects cotton fibre properties

Cotton fibre is the most important natural fibres for textile industry. To date, the mechanism that governs the development of fibre traits is largely unknown. In this study, we have characterized the function of a member of the actin depolymerizing factor (ADF) family in Gossypium hirsutum by down-regulation of the gene (designated as GhADF1 ) expression in the transgenic cotton plants. We observed that both the fibre length and strength of the GhADF1 -underexpressing plants increased as compared to the wild-type fibre, and transgenic fibres contained more abundant F-actin filaments in the cortical region of the cells. Moreover, the secondary cell wall of the transgenic fibre appeared thicker and the cellulose content was higher than that of the control fibre. Our results suggest that organization of actin cytoskeleton regulated by actin-associated proteins such as GhADF1 plays a critical role in the processes of elongation and secondary cell wall formation during fibre development. Additionally, our study provided a candidate intrinsic gene for the improvement of fibre traits via genetic engineering.     

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5'-flanking sequence

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5'-end Vim::cat constructs demonstrate that this repression is controlled by a 5'-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to +71) which do not contribute to vimentin's down-regulation during myogenesis.     

Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.     

DNA sequence variation in the promoter region of the VEGF gene impacts VEGF gene expression and maximal oxygen consumption

In its role as an endothelial cell proliferation and migration factor, vascular endothelial growth factor (VEGF) can affect peripheral circulation and therefore impact maximal oxygen consumption (Vo2 max). Because of the role of VEGF, and because variation in the VEGF gene has the ability to alter VEGF gene expression and VEGF protein level, we hypothesized that VEGF gene polymorphisms are related to VEGF gene expression in human myoblasts and Vo2 max before and after aerobic exercise training. We analyzed the effects of the VEGF -2578/-1154/-634 promoter region haplotype on VEGF gene expression by using a luciferase reporter assay in cultured human myoblasts and found that the AAG and CGC haplotypes resulted in significantly higher hypoxia-stimulated VEGF gene expression than the AGG and CGG haplotypes. Consistent with these results, we found that individuals with at least one copy of the AAG or CGC haplotype had higher Vo2 max before and after aerobic exercise training than did subjects with only the AGG and/or CGG haplotype. In conclusion, we found that VEGF -2578/-1154/-634 haplotype impacts VEGF gene expression in human myoblasts and is associated with Vo2 max. These results have potential implications for aerobic exercise training and may prove relevant in the study of pathological conditions that can be affected by angiogenesis, such as coronary artery disease and peripheral artery disease.     

Down-regulation of N-myristoyl transferase expression in human T-cell line CEM by human immunodeficiency virus type-1 infection

The present study focuses on the expression level of N-myristoyl transferase (NMT) in the course of human immunodeficiency virus type-1 (HIV-1) infection. HIV-1 structural proteins were gradually expressed during the process of infection of the human T-cell line CEM, whereas the expression levels of NMT subsequently decreased under the same conditions. In addition, the chronically HIV-1-infected T-cell line CEM/LAV-1 exhibited low expression levels of NMT. We hypothesize that the decrease in the expression level of NMT due to HIV-1 infection may be related to the virus' strategy that leads to its persistent replication.