Sequences of acetyl CoA carboxylase promoter for tumour necrosis factor action

Tumour necrosis factor (TNF) inhibits the accumulation of acetyl CoA carboxylase (ACC) mRNA by decreasing the rate of ACC gene transcription. The ACC mRNA species found in 30A5 cells are generated from promoter II and TNF inhibits the accumulation of class 2 type mRNAs. By using 5' deletion mutants of promoter II fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, the DNA mobility shift assay and the DNase I footprinting assay, the authors have identified the 30 bp from −389 to −359 as the TNF responsive element in promoter II. TNF treatment causes a decrease in the binding activity of nuclear protein(s) specific to the TNF responsive element. When the fragment containing the TNF responsive element was incorporated into the thymidine kinase promoter, the chimeric gene exhibited TNF induced inhibition of expression

Regulation of acetyl-coenzyme A carboxylase gene expression

Acetyl-Coenzyme A Carboxylase (acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) (ACC), is the biotin containing enzyme that catalyzes the rate-limiting step in the biogenesis of long-chain fatty acids. The regulatory mechanism of acetyl-CoA carboxylase at the gene level has been further established in the present studies. Under stimulated lipogenic conditions, ACC gene is activated and the forms of mRNA with different 5\sp\prime untranslated regions accumulate. This accumulation is the result of an increase in the rate of transcription. Multiple forms of ACC transcripts are generated as a result of differential splicing of two primary transcripts under the influence of two promoters, promoter I and promoter II. In 30A5 cells, ACC activity and mRNA levels increase during the differentiation process. The species of ACC mRNA accumulated are those transcribed from promoter II and there is little or no mRNA from promoter I. The conversion of 30A5 preadipocytes to adipocytes was much more accelerated by the cAMP pretreatment scheme or by the nutrient limitation scheme than by the classical procedure by 4 days. By examining chloramphenicol acetyltransferase (CAT) activities of stably transfected clones containing chimeric constructs between different fragments of the ACC promoter II and the promoterless CAT gene, we established that insulin induction on ACC gene expression and concomitant differentiation of 30A5 preadipocytes to adipocytes requires prior cAMP action and the presence of a specific promoter region which may contain cAMP responsive element(s). From DNase I footprinting analyses and gel retardation assays, we determined that the cis-element for cAMP action and binding of transcription factor(s) in 30A5 nuclear extracts is the AP-2 binding site ($-$108 to $-$100), GGGGCTGGG, in the ACC promoter II which also can be recognized by the purified human AP-2 protein

Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions. Keywords: adeno-associated virus, choroidal neovascularization, age-related macular generation, gene therap

Adeno-Associated Viral Vector-Mediated mTOR Inhibition by Short Hairpin RNA Suppresses Laser-Induced Choroidal Neovascularization

Choroidal neovascularization (CNV) is the defining characteristic feature of the wet subtype of age-related macular degeneration (AMD) and may result in irreversible blindness. Based on anti-vascular endothelial growth factor (anti-VEGF), the current therapeutic approaches to CNV are fraught with difficulties, and mammalian target of rapamycin (mTOR) has recently been proposed as a possible therapeutic target, although few studies have been conducted. Here, we show that a recombinant adeno-associated virus-delivered mTOR-inhibiting short hairpin RNA (rAAV-mTOR shRNA), which blocks the activity of both mTOR complex 1 and 2, represents a promising therapeutic approach for the treatment of CNV. Eight-week-old male C57/B6 mice were treated with the short hairpin RNA (shRNA) after generating CNV lesions in the eyes via laser photocoagulation. The recombinant adeno-associated virus (rAAV) delivery vehicle was able to effectively transduce cells in the inner retina, and significantly fewer inflammatory cells and less extensive CNV were observed in the animals treated with rAAV-mTOR shRNA when compared with control- and rAAV-scrambled shRNA-treated groups. Presumably related to the reduction of CNV, increased autophagy was detected in CNV lesions treated with rAAV-mTOR shRNA, whereas significantly fewer apoptotic cells detected in the outer nuclear layer around the CNV indicate that mTOR inhibition may also have neuroprotective effects. Taken together, these results demonstrate the therapeutic potential of mTOR inhibition, resulting from rAAV-mTOR shRNA activity, in the treatment of AMD-related CNV. Keywords: retinal neovascularization, choroidal neovascularization, adeno-associated virus, mTOR, RNA interference, mTOR shRNA, autophag

Secretion and cell volume regulation by salivary acinar cells from mice lacking expression of the Clcn3 Cl− channel gene

Salivary gland acinar cells shrink when Cl− currents are activated following cell swelling induced by exposure to a hypotonic solution or in response to calcium-mobilizing agonists. The molecular identity of the Cl− channel(s) in salivary cells involved in these processes is unknown, although ClC-3 has been implicated in several tissues as a cell-volume-sensitive Cl− channel. We found that cells isolated from mice with targeted disruption of the Clcn3 gene undergo regulatory volume decrease in a fashion similar to cells from wild-type littermates. Consistent with a normal regulatory volume decrease response, the magnitude and the kinetics of the swell-activated Cl− currents in cells from ClC-3-deficient mice were equivalent to those from wild-type mice. It has also been suggested that ClC-3 is activated by Ca2+-calmodulin-dependent protein kinase II; however, the magnitude of the Ca2+-dependent Cl− current was unchanged in the Clcn3−/- animals. In addition, we observed that ClC-3 appeared to be highly expressed in the smooth muscle cells of glandular blood vessels, suggesting a potential role for this channel in saliva production by regulating blood flow, yet the volume and ionic compositions of in vivo stimulated saliva from wild-type and null mutant animals were comparable. Finally, in some cells ClC-3 is an intracellular channel that is thought to be involved in vesicular acidification and secretion. Nevertheless, the protein content of saliva was unchanged in Clcn3−/- mice. Our results demonstrate that the ClC-3 Cl− channel is not a major regulator of acinar cell volume, nor is it essential for determining the secretion rate and composition of saliva